brANTI-DEspR MONOCLONAL ANTIBODY TARGETED THERAPY AND IMAGING FOR CANCER AND STROKE

ABSTRACT

Provided herein are novel compositions comprising anti-DEspR antibodies and fragments thereof derived from 6G8G7 and 7C5B2 anti-DEspR variant antibodies, including fully human, composite engineered human, humanized, monoclonal, and polyclonal anti-DEspR antibodies and fragments thereof, and methods of their use in a variety of therapeutic applications. The compositions comprising the anti-DEspR antibodies and fragments thereof described herein are useful in diagnostic and imaging methods, such as DEspR-targeted molecular imaging of angiogenesis, and for companion diagnostic and/or in vivo non-invasive imaging and/or assessments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/245,853 filed on Aug. 24, 2016, which claims benefit under 35U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 62/208,937filed on Aug. 24, 2015, the contents of which are herein incorporated byreference in their entirety.

GOVERNMENT SUPPORT

This invention was made with Government Support under Contract Nos.U54TR001012, HL058136, and AG032649 awarded by the National Institutesof Health. The Government has certain right in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 30, 2019, isnamed 701586-085062USD1_SL.txt and is 46,239 bytes in size.

FIELD OF THE INVENTION

This invention relates to monoclonal antibodies against the dualendothelin1NEGF-signal peptide receptor, DEspR, and their use astherapeutics in the inhibition of tumor initiation or progression orspread or recurrence and therapy resistance in cancer, and in theinhibition of microvascular leakiness or disruption, and microbleedssuch as occurs in, but not limited to, cancer and stroke, as well asdiagnostic agents and targeting agents for molecular imaging andtargeted delivery of other therapeutic agents.

BACKGROUND

Although targeted therapies have been tested, to date, there is noeffective therapy to stop therapy-resistant tumor recurrence orreseeding. Single targeted therapy that can stop tumor “reseeding” oftherapy-resistant tumors as seen in recurrent glioblastoma and inperitoneal carcinomatosis, such as occurs in pancreatic cancer, ovarianand gastric cancers provide a novel approach. Even if the primary tumorsresponded to current therapies, tumor recurrence usually results intherapy-resistant tumors—as seen in, for example, recurrentglioblastoma, pancreatic cancer, triple negative breast cancer (TNBC),and peritoneal carcinomatosis. Similarly, circulating tumor cells havebeen increasingly described, and serve as prognostic markers, but notherapy exists to inhibit them and prevent metastatic tumor initiation.Likewise, microvascular leakiness in tumors contributes to poor therapydelivery while facilitating egress of circulating tumor cells, but nosignificant therapy exists to address this. The basic rationale is thatthese cancer trends for recurrence can best be inhibited by asingle-agent that can simultaneously inhibit tumor initiation, therapyresistance, and microvessel leakiness.

In parallel, there is no therapy for patients with microvesselleakiness, disruption, and/or microbleeds in the brain that progress tomajor bleeds as seen in ischemic stroke patients (post-ischemichemorrhagic transformation or hemorrhagic conversion). In fact, a knowncomplication of the FDA-approved thrombolytic tissue-plasminogenactivator (TPA)-therapy for ischemic stroke when given late ishemorrhagic transformation. Once initiated, micro-to-macrobleedinitiaton-progression, or hemorrhagic transformation, leads to deatheven if the initating ischemic insult is resolved by current strokethrombolytic therapy. There too is no therapy for patients with brainmicrovessel leakiness, disruption, and/or microbleeds (detected on MRI)which are associated with subsequent pathologies, such as, but notlimited to stroke. The basic rationale is that microvessel leakiness,microbleeds and progression to hemorrhagic transformation or othermicrobleed-associated pathologies can best be stopped or prevented bypreventing development of microbleeds and their progression tomacrobleeds—collectively represented by microvacular leakiness, loss ofintegrity, and neutrophil-mediated injury.

SUMMARY OF THE INVENTION

Described herein are novel compositions comprising isolated antibodiesand antigen-binding fragments, including anti-DEspR antibodies andantigen-binding fragments thereof, derived from 6G8G7 and 7C5B2anti-DEspR variant antibodies, including humanized, fully human,composite engineered human, and deimmunized (T cell epitope-depleted)monoclonal anti-DEspR antibodies and antigen-binding fragments thereof,and methods of their use in a variety of applications, including,anti-angiogenesis therapies and anti-tumor cell invasiveness relevantfor treatment of cancer and/or metastasis and anti-angiogenesisapproaches relevant to treatment of those vascular diseases wherepathological angiogenesis plays a role in pathogenesis or progressionsuch as in carotid artery disease, stroke, ischemic hemorrhagictransformation, cerebral microbleeds, stroke, hemorrhagictransformation, vasa vasorum neovascularization, and vulnerable plaqueneovascularization.

Accordingly, provided herein, in some aspects is an isolated antibody orantigen-binding fragment thereof that has at least one of the followingfunctional characteristics:

-   -   a. an EC50 for binding to DEspR (dual endothelin/VEGF signal        peptide receptor) of 12 μg/ml or less;    -   b. an IC50 for inhibiting activated neutriphil survival or human        angiogenesis of 3.0 μg/ml or less; or    -   c. a K_(D) for binding DEspR of 2.5 μg/ml or less.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereof has anEC50 for binding to DEspR of 5 μg/ml or less.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereof has anEC50 for binding to DEspR of 30 nM or less.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereof, theIC50 for inhibiting activated neutriphil survival or human angiogenesisis 2.6 μg/ml or less.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereof,aK_(D) for binding DEspR is 1.5 μg/ml or less.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereof has atleast two of the functional characteristics.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereof hasall three of the functional characteristics.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereof is aneutralizing antibody or a DEspR antagonist.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereofspecifically binds to an epitope of DEspR of SEQ ID NO: 1 or SEQ ID NO:2.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereofcomprises one or more heavy and light chain complimentarity determiningregions (CDRs) selected from the group consisting of:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 8, SEQ ID NO: 15, or SEQ ID NO: 22;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 9, SEQ ID NO: 16, or SEQ ID NO: 23;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 28, SEQ ID NO: 35, SEQ ID NO: 42, or SEQ ID NO: 51;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 29, SEQ ID NO: 36, SEQ ID NO: 43, or SEQ ID NO: 52; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ ID NO: 53.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereofcomprises one or more heavy chain complimentarity determining regions(CDRs) selected from the group consisting of:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 8, SEQ ID NO: 15, or SEQ ID NO: 22;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 9, SEQ ID NO: 16, or SEQ ID NO: 23;    -   and one or more light chain CDRs selected from the group        consisting of:    -   a. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 28, SEQ ID NO: 35, SEQ ID NO: 42, or SEQ ID NO: 51;    -   b. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 29, SEQ ID NO: 36, SEQ ID NO: 43, or SEQ ID NO: 52; and    -   c. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ ID NO: 53.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereofcomprises:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 7;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 8;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 9;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 28;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 29; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 30.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereofcomprises:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 7;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 8;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 9;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 35;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 36; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 37.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereofcomprises:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 14;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 15;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 16;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 42;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 43; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 44.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereofcomprises:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 21;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 22;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 23;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 51;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 52; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 53.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereof is achimeric, humanized, or composite human antibody or dual antibody orantigen-binding fragment thereof.

In some embodiments of these aspects and all such aspects describedherein, the antibody fragment is a Fab fragment, a Fab′ fragment, a Fdfragment, a Fd′ fragment, a Fv fragment, a dAb fragment, a F(ab′)₂fragment, a single chain fragment, a diabody, or a linear antibody.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to an epitopeof DEspR (dual endothelin/VEGF signal peptide receptor) of SEQ ID NO: 1.

In some aspects, provided herein, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to an epitopeof DEspR (dual endothelin/VEGF signal peptide receptor) of SEQ ID NO: 2.

In some aspects, provided herein, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising one or more heavyand light chain complimentarity determining regions (CDRs) selected fromthe group consisting of:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 8, SEQ ID NO: 15, or SEQ ID NO: 22;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 9, SEQ ID NO: 16, or SEQ ID NO: 23;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 28, SEQ ID NO: 35, SEQ ID NO: 42, or SEQ ID NO: 51;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 29, SEQ ID NO: 36, SEQ ID NO: 43, or SEQ ID NO: 52; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ ID NO: 53.

In some embodiments of these aspects and all such aspects describedherein, the isolated anti-DEspR antibody or antigen-binding fragmentthereof comprises the heavy chain complimentarity determining regions(CDRs):

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 8, SEQ ID NO: 15, or SEQ ID NO: 22; and    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 9, SEQ ID NO: 16, or SEQ ID NO: 23

In some embodiments of these aspects and all such aspects describedherein, the isolated anti-DEspR antibody or antigen-binding fragmentthereof comprises the light chain complimentarity determining regions(CDRs):

-   -   a. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 28, SEQ ID NO: 35, SEQ ID NO: 42, or SEQ ID NO: 51;    -   b. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 29, SEQ ID NO: 36, SEQ ID NO: 43, or SEQ ID NO: 52; and    -   c. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ ID NO: 53.

In some embodiments of these aspects and all such aspects describedherein, the isolated anti-DEspR antibody or antigen-binding fragmentthereof comprises the complimentarity determining regions (CDRs):

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21:    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 8, SEQ ID NO: 15, or SEQ ID NO: 22;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 9, SEQ ID NO: 16, or SEQ ID NO: 23;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 28, SEQ ID NO: 35, SEQ ID NO: 42, or SEQ ID NO: 51;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 29, SEQ ID NO: 36, SEQ ID NO: 43, or SEQ ID NO: 52; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ ID NO: 53.

In some embodiments of these aspects and all such aspects describedherein, the isolated anti-DEspR antibody or antigen-binding fragmentthereof comprises a heavy chain having the amino acid sequence of SEQ IDNO: 6, SEQ ID NO: 13, or SEQ ID NO: 20.

In some embodiments of these aspects and all such aspects describedherein, the isolated anti-DEspR antibody or antigen-binding fragmentthereof comprises a light chain having the sequence of SEQ ID NO: 27,SEQ ID NO: 34, SEQ ID NO: 41, or SEQ ID NO: 50.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) comprising:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 7;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 8;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 9;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 28;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 29; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 30.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) comprising:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 7;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 8;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 9;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 35;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 36; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 37.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) comprising:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 7;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 8;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 9;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 42;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 43; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 44.

In some aspects, provided herein is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) comprising:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 7;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 8;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 9;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 51;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 52; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 53.

In some aspects, provided herein is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) comprising:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 14;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 15;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 16;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 28;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 29; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 30.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) comprising:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 14;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 15;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 16;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 35;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 36; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 37.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) comprising:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 14;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 15;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 16;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 42;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 43; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 44.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) comprising:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 14;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 15;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 16;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 51;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 52; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 53.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) comprising:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 21;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 22;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 23;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 28;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 29; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 30.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) comprising:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 21;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 22;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 23;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 35;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 36; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 37.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) comprising:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 21;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 22;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 23;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 42;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 43; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 44.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) comprising:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 21;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 22;    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 23;    -   d. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 51;    -   e. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 52; and    -   f. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 53.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising one or more heavychain complimentarity determining regions (CDRs) selected from the groupconsisting of:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 7;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 8; and    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 9.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising one or more heavychain complimentarity determining regions (CDRs) selected from the groupconsisting of:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO:14;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 15; and    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 16.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising one or more heavychain complimentarity determining regions (CDRs) selected from the groupconsisting of:

-   -   a. a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 21;    -   b. a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 22; and    -   c. a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 23.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising one or more lightchain complimentarity determining regions (CDRs) selected from the groupconsisting of:

-   -   a. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 28;    -   b. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 29; and    -   c. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 30.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising one or more lightchain complimentarity determining regions (CDRs) selected from the groupconsisting of:

-   -   a. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 35;    -   b. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 36; and    -   c. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 37.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising one or more lightchain complimentarity determining regions (CDRs) selected from the groupconsisting of:

-   -   a. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 42;    -   b. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 43; and    -   c. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 44.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising one or more lightchain complimentarity determining regions (CDRs) selected from the groupconsisting of:

-   -   a. a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 51;    -   b. a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 52; and    -   c. a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 53.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising a humanized variableheavy chain amino acid sequence of SEQ ID NO: 55.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising a humanized variablelight chain amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 59.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising a humanized variableheavy chain amino acid sequence of SEQ ID NO: 55, and a humanizedvariable light chain amino acid sequence of SEQ ID NO: 57 or SEQ ID NO:59.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising a humanized variableheavy chain IgG1 amino acid sequence of SEQ ID NO: 61.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising a humanized variableheavy chain IgG4 amino acid sequence of SEQ ID NO: 63.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising a humanized variablekappa light chain amino acid sequence of SEQ ID NO: 65.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising a humanized variableheavy chain IgG1 amino acid sequence of SEQ ID NO: 61 and a humanizedvariable kappa light chain amino acid sequence of SEQ ID NO: 65.

Provided herein, in some aspects, is an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds to DEspR (dualendothelin/VEGF signal peptide receptor) comprising a humanized variableheavy chain IgG4 amino acid sequence of SEQ ID NO: 63 and a humanizedvariable kappa light chain amino acid sequence of SEQ ID NO: 65.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody or antigen-binding fragment thereofspecifically binds to an epitope of SEQ ID NO: 1.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody or antigen-binding fragment thereofspecifically binds to an epitope of SEQ ID NO: 2.

In some embodiments of these aspects and all such aspects describedherein, the antibody is a chimeric, humanized, or composite humanantibody or dual antibody or antigen-binding fragment thereof.

In some embodiments of these aspects and all such aspects describedherein, the antibody fragment is a Fab fragment, a Fab′ fragment, a Fdfragment, a Fd′ fragment, a Fv fragment, a dAb fragment, a F(ab′)₂fragment, a single chain fragment, a diabody, or a linear antibody.

In some embodiments of these aspects and all such aspects describedherein, the isolated anti-DEspR antibody or antibody fragment thereoffurther comprises an agent conjugated to the anti-DEspR binding protein,antibody or antibody fragment, or antigen-binding portion thereofthereof to form an immunoconjugate specific for DEspR.

In some embodiments of these aspects and all such aspects describedherein, the agent conjugated to the binding protein, antibody orantibody fragment, or antigen-binding portion thereof thereof is achemotherapeutic agent, a toxin, a radioactive isotope, a smallmolecule, an siRNA, a nanoparticle, or a microbubble.

In some aspects, provided herein are pharmaceutical compositionscomprising any of the isolated anti-DEspR antibodies or antibodyfragments thereof described herein and a pharmaceutically acceptablecarrier.

In some aspects, provided herein is a method of inhibiting angiogenesisin a subject having a disease or disorder dependent or modulated byangiogenesis, the method comprising administering to a subject in needthereof a therapeutically effective amount of any of the pharmaceuticalcompositions described herein.

In some embodiments of these aspects and all such aspects describedherein, the disease or disorder dependent or modulated by angiogenesisis a cancer or a tumor.

In some embodiments of these aspects and all such aspects describedherein, the disease or disorder dependent or modulated by angiogenesisis selected from the group consisting of age-related maculardegeneration, carotid artery disease, diabetic retinopathy, rheumatoidarthritis, neurodegenerative disorder, Alzheimer's disease, obesity,endometriosis, psoriasis, atherosclerosis, ocular neovascularization,neovascular glaucoma, osteoporsosis, and restenosis.

In some aspects, provided herein is a method of inhibiting tumor cellinvasiveness in a subject having a cancer or a tumor, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of any of the pharmaceutical compositions describedherein.

In some embodiments of this aspect and all such aspects describedherein, the method further comprises the administration of one or morechemotherapeutic agents, angiogenesis inhibitors, cytotoxic agents,tumor-targeted therapies, immunotherapy, or anti-proliferative agents.

In some aspects, provided herein is a method of inhibiting tumor growthand reducing tumor size or tumor metastasis in a subject in need thereofby inhibiting DEspR expression and/or function in a cell, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of any of the pharmaceutical compositions describedherein.

In some embodiments of this aspect and all such aspects describedherein, the DEspR expression and/or function is inhibited in a tumorcell, a tumor initiating cell, a cancer stem-like cell, a cancer stemcell, a metastatic tumor cell, an endothelial progenitor cell, aninflammatory cell, a tumor stromal cell, a tumor vasculature cell, orany combination thereof.

In some embodiments of this aspect and all such aspects describedherein, the tumor vasculature cell is an endothelial cell, a pericyte, asmooth muscle cell, an adventitial cell, or any combination thereof.

In some aspects, provided herein is a method of inhibiting tumor therapyresistance, tumor initiation, and/or tumor recurrence by inhibitingDEspR expression and/or function in a cell, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of any of the pharmaceutical compositions described herein.

In some embodiments of this aspect and all such aspects describedherein, the DEspR expression and/or function is inhibited in a tumorcell, a tumor initiating cell, a cancer stem-like cell, a cancer stemcell, a metastatic tumor cell, or any combination thereof.

In some aspects, provided herein is a method of inhibiting cancerprogression through promotion of autophagy of a cancer cell byinhibiting DEspR expression and/or function in a tumor cell, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of any of the pharmaceutical compositions describedherein.

In some embodiments of this aspect and all such aspects describedherein, the DEspR expression and/or function is inhibited in a tumorcell, a tumor initiating cell, a cancer stem-like cell, a cancer stemcell, a metastatic tumor cell, or any combination thereof.

In some aspects, provided herein is a method of promoting autophagy or areduction in accumulation of intracellular noxious substances orpathogens by inhibiting DEspR expression and/or function, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of any of the pharmaceutical compositions describedherein.

In some embodiments of this aspect and all such aspects describedherein, the subject has Alzheimer's disease or Huntington's disease.

In some aspects, provided herein is a method of molecular imaging viatargeting DEspR, the method comprising administering an effective amountof any of the pharmaceutical compositions described herein conjugated toa targeting moiety, and determining the presence or absence of thepharmaceutical composition conjugated to the targeting moiety usingmolecular imaging.

In some embodiments of this aspect and all such aspects describedherein, the molecular imaging is contrast-enhanced ultrasound imaging,MRI (magnetic resonance imaging), near infrared imaging, orphotoacoustics imaging.

In some embodiments of this aspect and all such aspects describedherein, the targeting moiety is an antibody, a DEspR-binding peptideligand, a small molecule, a nanoparticle, a polymer, an aptamer, or anycombination thereof.

In some aspects, provided herein is a method for enhancing delivery of atherapeutic agent via DEspR-targeted sonoporation, the method comprisingdelivering an effective amount of any of the pharmaceutical compositionsdescribed herein and a therapeutic agent using targeted ultrasounddelivery to a subject in need thereof, wherein delivery of thetherapeutic agent is enhanced relative to delivering the therapeuticagent in the absence of the pharmaceutical composition.

In some embodiments of this aspect and all such aspects describedherein, the therapeutic agent is a chemotherapeutic agent, a smallmolecule, a peptide, or an aptamer.

In some aspects, provided herein is a method for reducing toxicity of atherapeutic agent via DEspR-targeted sonoporation, the method comprisingdelivering an effective amount of any of the pharmaceutical compositionsdescribed herein and a therapeutic agent using targeted ultrasounddelivery to a subject in need thereof, wherein toxicity of thetherapeutic agent is reduced relative to delivering the therapeuticagent in the absence of the pharmaceutical composition.

In some embodiments of this aspect and all such aspects describedherein, the therapeutic agent is a chemotherapeutic agent, a smallmolecule, a peptide, or an aptamer.

In some aspects, provided herein is a method for combiningDEspR-targeted molecular imaging and DEspR-targeted delivery of atherapeutic agent, the method comprising administering to a subject aneffective amount of a therapeutic agent and any of the pharmaceuticalcompositions described herein conjugated to a targeting moiety, anddetermining the presence or absence of the pharmaceutical compositionconjugated to the targeting moiety using molecular imaging.

In some embodiments of this aspect and all such aspects describedherein, the molecular imaging is contrast-enhanced ultrasound imaging,MRI (magnetic resonance imaging), near infrared imaging, orphotoacoustics imaging.

In some embodiments of this aspect and all such aspects describedherein, the therapeutic agent is a chemotherapeutic agent, a smallmolecule, a peptide, or an aptamer.

In some aspects, provided herein is a method of inhibiting tumorvascular leakiness by inhibiting DEspR expression and/or function in acell, the method comprising administering to a subject in need thereof atherapeutically effective amount of any of the pharmaceuticalcompositions described herein.

In some embodiments of this aspect and all such aspects describedherein, the DEspR expression and/or function is inhibited in a tumorcell, a tumor initiating cell, a cancer stem-like cell, a cancer stemcell, a metastatic tumor cell, an endothelial cell, an endotheialprogenitor cell, a stromal cell, an inflammatory cell, or anycombination thereof.

In some aspects, provided herein is a method of inhibiting peritonealcarcinomatosis by inhibiting DEspR expression and/or function in a cell,the method comprising administering to a subject in need thereof atherapeutically effective amount of any of the pharmaceuticalcompositions described herein.

In some embodiments of this aspect and all such aspects describedherein, the DEspR expression and/or function is inhibited in a tumorcell, a tumor initiating cell, a cancer stem-like cell, a cancer stemcell, a metastatic tumor cell, an endothelial cell, an endotheialprogenitor cell, a stromal cell, an inflammatory cell, a peritonealmesothelial cell, or any combination thereof.

A method of inhibiting microvascular leakiness, microvasculardisruption, microbleeds, or microvascular instability by inhibitingDEspR expression and/or function in a cell, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of any of the pharmaceutical compositions described herein.

In some embodiments of this aspect and all such aspects describedherein, the DEspR expression and/or function is inhibited in anendothelial cell, an endotheial progenitor cell, a pericyte, a vascularwall cell, a stromal cell, an inflammatory cell, or any combinationthereof.

In some embodiments of this aspect and all such aspects describedherein, the microvascular leakiness, microvascular disruption,microbleeds, or microvascular instability occurs in the brain.

In some aspects, provided herein is a method of inhibiting DEspRexpression and/or function using VEGFsp-26 peptide with or withoutmodifications that stabilize the peptide in vivo.

In some embodiments of this aspect and all such aspects describedherein, the VEGFsp-26 peptide comprises SEQ ID NO: 47.

In some embodiments of this aspect and all such aspects describedherein, the DEspR expression and/or function is inhibited in tumor cell,a tumor initiating cell, a cancer stem-like cell, a cancer stem cell, ametastatic tumor cell, an endothelial cell, an endotheial progenitorcell, a pericyte, a vascular wall cell, a stromal cell, an inflammatorycell, a peritoneal mesothelial cell, or any combination thereof.

In some aspects, provided herein is a method of stimulating DEspRexpression and/or function using a VEGFsp-17 peptide with or withoutmodifications that stabilize the peptide in vivo.

In some embodiments of this aspect and all such aspects describedherein, the VEGFsp-17 peptide comprises SEQ ID NO: 48.

In some embodiments of this aspect and all such aspects describedherein, wherein the DEspR expression and/or function is stimulated anendothelial cell, an endotheial progenitor cell, a pericyte, a vascularwall cell, a stromal cell, an inflammatory cell, or any combinationthereof.

Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology, andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011(ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway'sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor& Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4^(th) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties.

The term “DEspR binding protein construct” (or “DEspR binding protein”)refers to a polypeptide that specifically binds to DEspR and is anIg-like protein comprising one or more of the antigen binding portionsdescribed herein linked to a linker or an immunoglobulin constantdomain. A binding protein can be a dual variable domain (DVD-Ig) bindingprotein. A “linker polypeptide” comprises two or more amino acidresidues joined by peptide bonds and are used to link one or moreantigen binding portions. Such linker polypeptides are well known in theart (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123). An immunoglobulinconstant domain refers to a heavy or light chain constant domain. HumanIgG heavy chain and light chain constant domain amino acid sequences areknown in the art, (e.g., see SEQ ID NO: 197, 198, 199 and 200 of USApplication 2016/0200813, which is incorporated herein in its entiretyby reference for representative examples). In various embodiments, thebinding proteins and antibodies disclosed herein can comprise any of theconstant domains of SEQ ID NO: 197, 198, 199 and 200 of US Application2016/0200813.

The term “antibody” broadly refers to any immunoglobulin (Ig) moleculeand immunologically active portions of immunoglobulin molecules (i.e.,molecules that contain an antigen binding site that immunospecificallybind an antigen) comprised of four polypeptide chains, two heavy (H)chains and two light (L) chains, or any functional fragment, mutant,variant, or derivation thereof, which retains the essential epitopebinding features of an Ig molecule. Such mutant, variant, or derivativeantibody formats are known in the art. Nonlimiting embodiments of whichare discussed below, and include but are not limited to a variety offorms, including full length antibodies and antigen-binding portionsthereof; including, for example, an immunoglobulin molecule, amonoclonal antibody, a chimeric antibody, a CDR-grafted antibody, ahuman antibody, a humanized antibody, a single chain antibody, a Fab, aF(ab′), a F(ab′)2, a Fv antibody, fragments produced by a Fab expressionlibrary, a disulfide linked Fv, a scFv, a single domain antibody (dAb),a diabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, a functionally activeepitope-binding fragment thereof, bifunctional hybrid antibodies (e.g.,Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains(e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883(1988) and Bird et al., Science 242, 423-426 (1988), which areincorporated herein by reference) and/or antigen-binding fragments ofany of the above (See, generally, Hood et al., Immunology, Benjamin,N.Y., 2ND ed. (1984), Harlow and Lane, Antibodies. A Laboratory Manual,Cold Spring Harbor Laboratory (1988) and Hunkapiller and Hood, Nature,323, 15-16 (1986), which are incorporated herein by reference).Antibodies also refer to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain antigen or target binding sites or “antigen-binding fragments.”The antibody or immunoglobulin molecules described herein can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule, as isunderstood by one of skill in the art. Furthermore, in humans, the lightchain can be a kappa chain or a lambda chain.

In a full-length antibody, each heavy chain is comprised of a heavychain variable domain (abbreviated herein as HCVR or V_(H)) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains: CH1, CH2, and CH3. Each light chain is comprised of alight chain variable domain (abbreviated herein LCVR as V_(L)) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. This structure is well-known to those skilled in the art. Thechains are usually linked to one another via disulfide bonds.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain, which may be generated by papain digestionof an intact antibody. The Fc region may be a native sequence Fc regionor a variant Fc region. The Fc region of an immunoglobulin generallycomprises two constant domains, a CH2 domain, and a CH3 domain, andoptionally comprises a CH4 domain. Replacements of amino acid residuesin the Fc portion to alter antibody effector function are known in theart (U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of anantibody mediates several important effector functions, for example,cytokine induction, ADCC, phagocytosis, complement dependentcytotoxicity (CDC), and half-life/clearance rate of antibody andantigen-antibody complexes. In some cases these effector functions aredesirable for therapeutic antibody but in other cases might beunnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to Fc.gamma.Rs and complement C1q,respectively. Neonatal Fc receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies. In still anotherembodiment at least one amino acid residue is replaced in the constantregion of the antibody, for example the Fc region of the antibody, suchthat effector functions of the antibody are altered.

The term “antigen-binding portion” of an antibody refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., DEspR). Antigen-binding functions of an antibody canbe performed by fragments of a full-length antibody. Such antibodyfragment embodiments may also be incorporated in bispecific, dualspecific, or multi-specific formats such as a dual variable domain(DVD-Ig) format; specifically binding to two or more different antigens(e.g., DEspR and a different antigen molecule). Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL, and CH1 domains; (ii) a F(ab′)² fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al. (1989)Nature, 341: 544-546; PCT Publication No. WO 90/05144), which comprisesa single variable domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); see,for example, Bird et al. (1988) Science 242: 423-426; and Huston et al.(1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see, for example, Holliger etal. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak (1994)Structure 2: 1121-1123); Kontermann and Dubel eds., AntibodyEngineering, Springer-Verlag, N.Y. (2001), p. 790 (ISBN 3-540-41354-5).In addition single chain antibodies also include “linear antibodies”comprising a pair of tandem Fv segments (VH—CH1-VH—CH1) which, togetherwith complementary light chain polypeptides, form a pair of antigenbinding regions (Zapata et al. (1995) Protein Eng. 8(10): 1057-1062; andU.S. Pat. No. 5,641,870).

An immunoglobulin constant (C) domain refers to a heavy (C_(H)) or light(C_(L)) chain constant domain. Murine and human IgG heavy chain andlight chain constant domain amino acid sequences are known in the art.

As used herein, an “anti-DEspR antibody” refers to an antibody thatbinds to DEspR with sufficient affinity and specificity. The antibodyselected will normally have a binding affinity for DEspR, for example,the antibody can bind human DEspR protein with a K_(D) value between10⁻⁵ M to 10⁻¹⁰ M.

A DEspR binding protein, antibody, or antigen-binding portion thereof,may be part of a larger immunoadhesion molecule, formed by covalent ornoncovalent association of the antibody antigen-binding portion with oneor more other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov et al. (1995) Human Antibod.Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide anda C-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058).Antibody portions, such as Fab and F(ab′).sub.2 fragments, can beprepared from whole antibodies using conventional techniques, such aspapain or pepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antigen-binding portions thereof, and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques. ADEspR binding protein, such as an antigen-binding portion of an antibodymay also be part of a dual variable domain (DVD-Ig).

As used herein, the term “target” refers to a biological molecule (e.g.,peptide, polypeptide, protein, lipid, carbohydrate) to which apolypeptide domain which has a binding site can selectively bind. Thetarget can be, for example, an intracellular target (e.g., anintracellular protein target) or a cell surface target (e.g., a membraneprotein, a receptor protein). Preferably, a target is a cell surfacetarget, such as a cell surface protein.

As described herein, an “antigen” is a molecule that is bound by abinding site on a polypeptide agent, such as a binding protein, anantibody or antibody fragment, or antigen-binding fragment thereof.Typically, antigens are bound by antibody ligands and are capable ofraising an antibody response in vivo. An antigen can be a polypeptide,protein, nucleic acid or other molecule. In the case of conventionalantibodies and fragments thereof, the antibody binding site as definedby the variable loops (L1, L2, L3 and H1, H2, H3) is capable of bindingto the antigen. The term “antigenic determinant” refers to an epitope onthe antigen recognized by an antigen-binding molecule, and moreparticularly, by the antigen-binding site of said molecule.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound by abinding protein. An epitope may be determined by obtaining an X-raycrystal structure of an antibody:antigen complex and determining whichresidues on the antigen (DEspR) are within a specified distance ofresidues on the antibody of interest, wherein the specified distance is,5 Å or less, e.g., 5 Å, 4 Å, 3 Å, 2 Å, 1 Å or any distance in between.In some embodiments, an “epitope” can be formed on a polypeptide (e.g.,DEspR) both from contiguous amino acids, or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents, whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5, about 9, or about 8-10 aminoacids in a unique spatial conformation. An “epitope” includes the unitof structure conventionally bound by an immunoglobulin V_(H)/V_(L) pair.Epitopes define the minimum binding site for an antibody, and thusrepresent the target of specificity of an antibody. In the case of asingle domain antibody, an epitope represents the unit of structurebound by a variable domain in isolation. The terms “antigenicdeterminant” and “epitope” can also be used interchangeably herein. Incertain embodiments, epitope determinants include chemically activesurface groupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. In some embodiments, an epitope comprises of 8 or morecontiguous or non-contiguous amino acid residues in the DEspR sequencein which at least 50%, 70% or 85% of the residues are within thespecified distance of the antibody or binding protein in the X-raycrystal structure.

The terms “specificity” or “specific for” refers to the number ofdifferent types of antigens or antigenic determinants to which a bindingprotein, antibody or antibody fragment, or antigen-binding portionthereof thereof as described herein can bind. The specificity of abinding protein, antibody or antibody fragment, or antigen-bindingportion thereof thereof can be determined based on affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation (K_(D)) of an antigen with an antigen-binding protein, is ameasure of the binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein, such as a bindingprotein, antibody or antibody fragment, or antigen-binding portionthereof thereof: the lesser the value of the K_(D), the stronger thebinding strength between an antigenic determinant and theantigen-binding molecule. Alternatively, the affinity can also beexpressed as the affinity constant (K_(A)), which is 1/K_(D)). As willbe clear to the skilled person, affinity can be determined in a mannerknown per se, depending on the specific antigen of interest.Accordingly, a binding protein, antibody or antibody fragment, orantigen-binding portion thereof thereof as defined herein is said to be“specific for” a first target or antigen compared to a second target orantigen when it binds to the first antigen with an affinity (asdescribed above, and suitably expressed, for example as a K_(D) value)that is at least 10 times, such as at least 100 times, and preferably atleast 1000 times, and up to 10000 times or more better than the affinitywith which said amino acid sequence or polypeptide binds to anothertarget or polypeptide.

Accordingly, as used herein, “selectively binds” or “specifically binds”or “specific binding” in reference to the interaction of an antibody, orantibody fragment thereof, or a binding protein described herein, meansthat the interaction is dependent upon the presence of a particularstructure (e.g., an antigenic determinant or epitope or target) on thechemical species; for example, an antibody recognizes and binds to aspecific protein structure rather than to proteins generally. If anantibody is specific for epitope “A”, the presence of a moleculecontaining epitope A (or free, unlabeled A), in a reaction containinglabeled “A” and the antibody, will reduce the amount of labeled A boundto the antibody. In certain embodiments, a binding protein or antibodyor antigen-binding fragment thereof that specifically binds to anantigen binds to that antigen with a K_(D) greater than 10⁻⁶ M, 10⁻⁷ M,10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻M, 10⁻¹² M, 10⁻¹³ M, 10⁻¹⁴ M. In otherembodiments, a binding protein or antibody or antigen binding fragmentthereof that specifically binds to an antigen binds to that antigen witha K_(D) between 10⁻⁶ and 10⁻⁷M, 10⁻⁶ and 10⁻⁸ M, 10⁻⁶ and 10⁻⁹ M, 10⁻⁶and 10¹⁰ M, 10⁻⁶ and 10⁻¹¹ M, 10⁻⁶ and 10⁻¹²M, 10⁻⁶ and 10⁻¹³ M, and10⁻⁶ and 10⁻¹⁴ M, 10⁻⁹ and 10⁻¹⁰ M, 10⁻⁹ and 10⁻¹¹ M, 10⁻⁹ and 10⁻¹² M,10⁻⁹ and 10⁻¹³ M, 10⁻⁹ and 10⁻¹⁴M. In some embodiments, a bindingprotein or antibody or antigen-binding fragment thereof binds to anepitope, with a K_(D) 10⁻⁵ M (10000 nM) or less, e.g., 10⁻⁶ M, 10⁻⁷ M,10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, or less. Specific binding canbe influenced by, for example, the affinity and avidity of thepolypeptide agent and the concentration of polypeptide agent. The personof ordinary skill in the art can determine appropriate conditions underwhich the polypeptide agents described herein selectively bind thetargets using any suitable methods, such as titration of a polypeptideagent in a suitable cell binding assay. In certain embodiments, abinding protein or antibody or antigen-binding fragment thereof is saidto “specifically bind” an antigen when it preferentially recognizes itstarget antigen in a complex mixture of proteins and/or macromolecules.Binding proteins, antibodies or antigen-binding fragments that bind tothe same or similar epitopes will likely cross-compete (one prevents thebinding or modulating effect of the other). Cross-competition, however,can occur even without epitope overlap, e.g., if epitopes are adjacentin three-dimensional space and/or due to steric hindrance.

Avidity is the measure of the strength of binding between anantigen-binding molecule (such as a binding protein, antibody orantibody fragment, or antigen-binding portion thereof thereof describedherein) and the pertinent antigen. Avidity is related to both theaffinity between an antigenic determinant and its antigen binding siteon the antigen-binding molecule, and the number of pertinent bindingsites present on the antigen-binding molecule. Typically,antigen-binding proteins (such as a binding protein, antibody orantibody fragment, or antigen-binding portion thereof thereof describedherein) will bind to their cognate or specific antigen with adissociation constant (K_(D) of 10⁻⁵ to 10⁻¹² moles/liter or less, andpreferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably 10⁻⁸ to10⁻¹² moles/liter (i.e., with an association constant (K_(A)) of 10⁵ to10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or moreand more preferably 10⁸ to 10¹² liter/moles). Any K_(D) value greaterthan 10⁻⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹) is generallyconsidered to indicate non-specific binding. The K_(D) for biologicalinteractions which are considered meaningful (e.g., specific) aretypically in the range of 10⁻¹⁰ M (0.1 nM) to 10⁻⁵ M (10000 nM). Thestronger an interaction is, the lower is its K_(D). Preferably, abinding site on a binding protein, antibody or antibody fragment, orantigen-binding portion thereof thereof described herein will bind tothe desired antigen with an affinity less than 500 nM, preferably lessthan 200 nM, more preferably less than 10 nM, such as less than 500 pM.Specific binding of an antigen-binding protein to an antigen orantigenic determinant can be determined in any suitable manner known perse, including, for example, Scatchard analysis and/or competitivebinding assays, such as radioimmunoassays (RIA), enzyme immunoassays(EIA) and sandwich competition assays, and the different variantsthereof known per se in the art; as well as other techniques asmentioned herein.

In some embodments, an anti-DEspR binding protein, antibody or antibodyfragment, or antigen-binding portion thereof thereof described hereinbinds to DEspR, with a K_(D) 10⁻⁵ M (10000 nM) or less, e.g., 10⁻⁶ M,10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹M, 10¹² M, or less. In someembodiments, where an antibody or antigen-binding fragment thereof isdirected to an epitope or antigenic peptide, the antibody orantigen-binding fragment thereof can be referred to, for example, as anantibody or antigen-binding fragment thereof directed to or specific forSEQ ID NO: 1 or SEQ ID NO: 2.

The term “K_(on)” (also “Kon”, “kon”), as used herein, is intended torefer to the on rate constant for association of a binding protein,antibody or antigen-binding fragment to an antigen to form anassociation complex, e.g., binding protein/antigen complex, as is knownin the art. The “K_(on)” also is known by the terms “association rateconstant”, or “ka”, as used interchangeably herein. This value indicatesthe binding rate of a binding protein to its target antigen or the rateof complex formation between an antibody and antigen as is shown by theequation below:

Binding protein (“Ab”)+Antigen (“Ag”)→Ab-Ag.

The term “K_(off)” (also “Koff”, “koff”), as used herein, is intended torefer to the off rate constant for dissociation, or “dissociation rateconstant”, of a binding protein, antibody or antigen-binding fragmentfrom an association complex (e.g., a binding protein/antigen complex) asis known in the art. This value indicates the dissociation rate of anantibody from its target antigen or separation of Ab-Ag complex overtime into free binding protein and antigen as shown by the equationbelow:

Ab+Ag←Ab-Ag.

The term “K_(D)” (also “K_(d)”), as used herein, is intended to refer tothe “equilibrium dissociation constant”, and refers to the valueobtained in a titration measurement at equilibrium, or by dividing thedissociation rate constant (Koff) by the association rate constant(Kon). The association rate constant (Kon), the dissociation rateconstant (Koff), and the equilibrium dissociation constant (K are usedto represent the binding affinity of a binding protein to an antigen.Methods for determining association and dissociation rate constants arewell known in the art. Using fluorescence-based techniques offers highsensitivity and the ability to examine samples in physiological buffersat equilibrium. Other experimental approaches and instruments such as aBIAcore.®. (biomolecular interaction analysis) assay can be used (e.g.,instrument available from BIAcore International AB, a GE Healthcarecompany, Uppsala, Sweden). Additionally, a KinExA.®. (Kinetic ExclusionAssay) assay, available from Sapidyne Instruments (Boise, Id.) can alsobe used.

The term “antibody fragment,” or “antigen-binding fragment” as usedherein, refer to a protein fragment that comprises only a portion of anintact antibody, generally including an antigen binding site of theintact antibody and thus retaining the ability to bind antigen. Examplesof antibody fragments encompassed by the present definition include: (i)the Fab fragment, having V_(L), C_(L), V_(H) and C_(H)1 domains; (ii)the Fab′ fragment, which is a Fab fragment having one or more cysteineresidues at the C-terminus of the C_(H)1 domain; (iii) the Fd fragmenthaving V_(H) and C_(H)1 domains; (iv) the Fd′ fragment having V_(H) andC_(H)1 domains and one or more cysteine residues at the C-terminus ofthe CH1 domain; (v) the Fv fragment having the V_(L) and V_(H) domainsof a single arm of an antibody; (vi) the dAb fragment (Ward et al.,Nature 341, 544-546 (1989)) which consists of a V_(H) domain; (vii)isolated CDR regions; (viii) F(ab′)₂ fragments, a bivalent fragmentincluding two Fab′ fragments linked by a disulphide bridge at the hingeregion; (ix) single chain antibody molecules (e.g., single chain Fv;scFv) (Bird et al., Science 242:423-426 (1988); and Huston et al., PNAS(USA) 85:5879-5883 (1988)); (x) “diabodies” with two antigen bindingsites, comprising a heavy chain variable domain (VH) connected to alight chain variable domain (V_(L)) in the same polypeptide chain (see,e.g., EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993)); (xi) “linear antibodies” comprising apair of tandem Fd segments (V_(H)—C_(H)1-V_(H)—C_(H)1) which, togetherwith complementary light chain polypeptides, form a pair of antigenbinding regions (Zapata et al. Protein Eng. 8(10):1057-1062 (1995); andU.S. Pat. No. 5,641,870).

The term anti-DEspR “antigen-binding fragment” refers to a proteinfragment that comprises at least an antigen binding site of the intactantibody and thus retains the ability to bind a DEspR antigen orepitope. Non-limiting examples of antibody fragments encompassed by theterm antigen-binding fragment include: (i) the Fab fragment, havingV_(L), C_(L), V_(H) and C_(H) 1 domains; (ii) the Fab′ fragment, whichis a Fab fragment having one or more cysteine residues at the C-terminusof the C_(H)1 domain; (iii) the Fd fragment having V_(H) and C_(H) 1domains; (iv) the Fd′ fragment having V_(H) and C_(H)1 domains and oneor more cysteine residues at the C-terminus of the CH1 domain; (v) theFv fragment having the V_(L) and V_(H) domains of a single arm of anantibody; (vi) the dAb fragment (Ward et al., Nature 341, 544-546(1989)) which consists of a V_(H) domain; (vii) isolated CDR regions;(viii) F(ab′)₂ fragments, a bivalent fragment including two Fab′fragments linked by a disulphide bridge at the hinge region; (ix) singlechain antibody molecules (e.g., single chain Fv; scFv) (Bird et al.,Science 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883(1988)); (x) “diabodies” with two antigen binding sites, comprising aheavy chain variable domain (V_(H)) connected to a light chain variabledomain (V_(L)) in the same polypeptide chain (see, e.g., EP 404,097; WO93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993)); (xi) “linear antibodies” comprising a pair of tandem Fdsegments (V_(H)—C_(H)1-V_(H)—C_(H)1) which, together with complementarylight chain polypeptides, form a pair of antigen binding regions (Zapataet al. Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No.5,641,870).

An “Fv” fragment is an antibody fragment which contains a completeantigen recognition and binding site. This region consists of a dimer ofone heavy and one light chain variable domain in tight association,which can be covalent in nature, for example in scFv. It is in thisconfiguration that the three CDRS of each variable domain interact todefine an antigen binding site on the surface of the V_(H)—V_(L) dimer.Collectively, the six CDRS or a subset thereof confer antigen bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRS specific for an antigen) hasthe ability to recognize and bind antigen, although usually at a loweraffinity than the entire binding site.

The “Fab” fragment contains a variable and constant domain of the lightchain and a variable domain and the first constant domain (C_(H)1) ofthe heavy chain. F(ab′)₂ antibody fragments comprise a pair of Fabfragments which are generally covalently linked near their carboxytermini by hinge cysteines between them. Other chemical couplings ofantibody fragments are also known in the art.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains, which enablesthe scFv to form the desired structure for antigen binding For a reviewof scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term “monoclonal antibody” or “mAb” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that canbe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigen. Furthermore, in contrast topolyclonal antibody preparations that typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the invention can bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or can be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” can also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) or Marks et al., J. Mol.Biol. 222:581-597 (1991), for example. A monoclonal antibody can be ofany species, including, but not limited to, mouse, rat, goat, rabbit,and human monoclonal antibodies. Various methods for making monoclonalantibodies specific for DEspR as described herein are available in theart. For example, the monoclonal antibodies can be made using thehybridoma method first described by Kohler et al., Nature, 256:495(1975), or by recombinant DNA methods (U.S. Pat. No. 4,816,567).“Monoclonal antibodies” can also be isolated from or produced usingphage antibody libraries using the techniques originally described inClackson et al., Nature 352:624-628 (1991), Marks et al., J. Mol. Biol.222:581-597 (1991), McCafferty et al., Nature, 348:552-554 (1990), Markset al., Bio/Technology, 10:779-783 (1992)), Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993), and techniques known to those ofordinary skill in the art.

The term “human antibody” includes antibodies having variable andconstant regions derived from human germline immunoglobulin sequences.The human antibodies of the disclosure may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “human antibody” does not include antibodies in whichCDR sequences derived from the germline of another mammalian species,such as a mouse, have been grafted onto human framework sequences.

The term “recombinant human antibody” includes all human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes, orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences.In certain embodiments, however, such recombinant human antibodies aresubjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis) and thus theamino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the humanantibody germline repertoire in vivo.

The term “chimeric antibody” refers to antibodies that comprise heavyand light chain variable domain sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable domains linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies that comprise heavyand light chain variable domain sequences from one species but in whichthe sequences of one or more of the CDR regions of VH and/or VL arereplaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable domains in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “CDR” refers to the complementarity determining region withinantibody variable sequences. There are three CDRs in each of thevariable domains of the heavy chain and the light chain, which aredesignated CDR1, CDR2 and CDR3, for each of the variable domains. Theterm “CDR set” as used herein refers to a group of three CDRS that occurin a single variable domain capable of binding the antigen. The exactboundaries of these CDRs have been defined differently according todifferent systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest, National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable domainof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRS may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia et al. (1987) J. Mol. Biol. 196: 901-917; and Chothiaet al. (1989) Nature 342: 877-883) found that certain sub-portionswithin Kabat CDRS adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2, and L3 or H1, H2, and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRS have beendescribed by Padlan et al. ((1995) FASEB J. 9:133-139) and MacCallum etal. ((1996) J. Mol. Biol. 262(5):732-745). Still other CDR boundarydefinitions may not strictly follow one of the above systems, but willnonetheless overlap with the Kabat CDRs, although they may be shortenedor lengthened in light of prediction or experimental findings thatparticular residues or groups of residues or even entire CDRs do notsignificantly impact antigen binding. The methods used herein mayutilize CDRS defined according to any of these systems, althoughexemplary embodiments use Kabat or Chothia defined CDRs.

The terms “Kabat numbering”, “Kabat definitions”, and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e., hypervariable) than other amino acid residues inthe heavy and light chain variable domains of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci.190:382-391; and Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, which is also available onthe world wide web, and is expressly incorporated herein in its entiretyby reference. The “EU index as in Kabat” refers to the residue numberingof the human IgG1 EU antibody. As used herein, “Kabat sequencenumbering” refers to numbering of the sequence encoding a variableregion according to the EU index as in Kabat. In some embodiments, IMGT(INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM) numbering of variableregions can also be used, which is the numbering of the residues in animmunoglobulin variable heavy or light chain according to the methods ofthe IIMGT, as described in Lefranc, M.-P., “The IMGT unique numberingfor immunoglobulins, T cell Receptors and Ig-like domains”, TheImmunologist, 7, 132-136 (1999), and is expressly incorporated herein inits entirety by reference. As used herein, “IMGT sequence numbering”refers to numbering of the sequence encoding a variable region accordingto the IMGT. For the heavy chain variable domain, the hypervariableregion ranges from amino acid positions 31 to 35 for CDR1, amino acidpositions 50 to 65 for CDR2, and amino acid positions 95 to 102 forCDR3. For the light chain variable domain, the hypervariable regionranges from amino acid positions 24 to 34 for CDR1, amino acid positions50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

The growth and analysis of extensive public databases of amino acidsequences of variable heavy and light regions over the past twenty yearshave led to the understanding of the typical boundaries betweenframework regions (FR) and CDR sequences within variable regionsequences and enabled persons skilled in this art to accuratelydetermine the CDRs according to Kabat numbering, Chothia numbering, orother systems. See, e.g., Martin, “Protein Sequence and StructureAnalysis of Antibody Variable Domains,” Chapter 31, In AntibodyEngineering, (Kontermann and Dubel, eds.) (Springer-Verlag, Berlin,2001), especially pages 432-433. A useful method of determining theamino acid sequences of Kabat CDRs within the amino acid sequences ofvariable heavy (VH) and variable light (VL) regions are known in theart, e.g., as follows:

To identify a CDR-L1 amino acid sequence: Starts approximately 24 aminoacid residues from the amino terminus of the VL region; Residue beforethe CDR-L1 sequence is always cysteine (C); Residue after the CDR-L1sequence is always a tryptophan (W) residue, typically Trp-Tyr-Gln(W-Y-Q), but also Trp-Leu-Gln (W-L-Q), Trp-Phe-Gln (W-F-Q), andTrp-Tyr-Leu (W-Y-L); Length is typically 10 to 17 amino acid residues.To identify a CDR-L2 amino acid sequence: Starts always 16 residuesafter the end of CDR-L1; Residues before the CDR-L2 sequence aregenerally Ile-Tyr (I-Y), but also Val-Tyr (V-Y), Ile-Lys (1-K), andIle-Phe (1-F); Length is always 7 amino acid residues. To identify aCDR-L3 amino acid sequence: Starts always 33 amino acids after the endof CDR-L2; Residue before the CDR-L3 amino acid sequence is always acysteine (C); Residues after the CDR-L3 sequence are alwaysPhe-Gly-X-Gly (F-G-X-G) (SEQ ID NO: 68), where X is any amino acid;Length is typically 7 to 11 amino acid residues.

To identify a CDR-H1 amino acid sequence: Starts approximately 31 aminoacid residues from amino terminus of VH region and always 9 residuesafter a cysteine (C); Residues before the CDR-H1 sequence are alwaysCys-X-X-X-X-X-X-X-X (SEQ ID NO: 69), where X is any amino acid; Residueafter CDR-H1 sequence is always a Trp (W), typically Trp-Val (W-V), butalso Trp-Ile (W-I), and Trp-Ala (W-A); Length is typically 5 to 7 aminoacid residues. To identify a CDR-H2 amino acid sequence: Starts always15 amino acid residues after the end of CDR-H1; Residues before CDR-H2sequence are typically Leu-Glu-Trp-Ile-Gly (L-E-W-I-G) (SEQ ID NO: 70),but other variations also; Residues after CDR-H2 sequence areLys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala(K/R-L/I/V/F/T/A-T/S/I/A); Length is typically 16 to 19 amino acidresidues. To identify a CDR-H3 amino acid sequence: Starts always 33amino acid residues after the end of CDR-H2 and always 3 after acysteine (C)′ Residues before the CDR-H3 sequence are always Cys-X-X(C-X-X), where X is any amino acid, typically Cys-Ala-Arg (C-A-R);Residues after the CDR-H3 sequence are always Trp-Gly-X-Gly (W-G-X-G)(SEQ ID NO: 71), where X is any amino acid; Length is typically 3 to 25amino acid residues.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. ((1987) J. Mol. Biol. 196: 901-917); andChothia et al. ((1992) J. Mol. Biol. 227: 799-817), both areincorporated herein by reference). According to Chothia et al., criticalportions of the CDRs of many antibodies have nearly identical peptidebackbone confirmations despite great diversity at the level of aminoacid sequence. Each canonical structure specifies primarily a set ofpeptide backbone torsion angles for a contiguous segment of amino acidresidues forming a loop.

As used herein, “antibody variable domain” refers to the portions of thelight and heavy chains of antibody molecules that include amino acidsequences of Complementarity Determining Regions (CDRs; i.e., CDR1,CDR2, and CDR3), and Framework Regions (FRs). Each heavy chain iscomposed of a variable region of the heavy chain (V_(H) refers to thevariable domain of the heavy chain) and a constant region of said heavychain. The heavy chain constant region consists of three domains CH1,CH2 and CH3. Each light chain is composed of a variable region of saidlight chain (V_(L) refers to the variable domain of the light chain) anda constant region of the light chain. The light chain constant regionconsists of a CL domain. The VH and VL regions can be further dividedinto hypervariable regions referred to as complementarity-determiningregions (CDRs) and interspersed with conserved regions referred to asframework regions (FR). Each VH and VL region thus consists of threeCDRS and four FRs that are arranged from the N terminus to the Cterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.This structure is well known to those skilled in the art. According tothe methods used herein, the amino acid positions assigned to CDRS andFRs can be defined according to Kabat (Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991)). Amino acid numbering of antibodies or antigen bindingfragments is also according to that of Kabat.

As used herein, the term “Complementarity Determining Regions” (“CDRs”),i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding. Each variable domain typically has three CDR regions identifiedas CDR1, CDR2 and CDR3. Each complementarity determining region cancomprise amino acid residues from a “complementarity determining region”as defined by Kabat (i.e., about residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (i.e., about residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In someinstances, a complementarity determining region can include amino acidsfrom both a CDR region defined according to Kabat and a hypervariableloop. For example, the CDRH1 of the heavy chain of antibody 4D5 includesamino acids 26 to 35.

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and of the light chain, whichare designated CDR 1, CDR2 and CDR3, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region capable of binding the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Eachcomplementarity determining region may comprise amino acid residues froma “complementarity determining region” as defined by Kabat (i.e. aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chainvariable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavychain variable domain; Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)) and/or those residues from a“hypervariable loop” (i.e. about residues 26-32 (L1), 50-52 (L2) and91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2)and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). In some instances, a complementaritydetermining region can include amino acids from both a CDR regiondefined according to Kabat and a hypervariable loop. For example, theCDRH1 of the human heavy chain of antibody 4D5 includes amino acids 26to 35. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol, 196:901-917(1987) and Chothia et al., Nature 342:877-883 (-1989)) found thatcertain sub-portions within Kabat CDRs adopt nearly identical peptidebackbone conformations, in spite of great diversity at the level ofamino acid sequence. These sub-portions were designated as L1, L2 and L3or H1, H2 and H3 where the “L” and the “H” designates the light chainand the heavy chains regions, respectively. These regions may bereferred to as Chothia CDRs, which have boundaries that overlap withKabat CDRs. Other boundaries defining CDRs overlapping with the KabatCDRs have been described by Padlan (FASEB). 9:133-139 (1995)) andMacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundarydefinitions may not strictly follow one of the above systems, but willnonetheless overlap with the Kabat CDRs, although they may be shortenedor lengthened in light of prediction or experimental findings thatparticular residues or groups of residues or even entire CDRS do notsignificantly impact antigen binding. The methods used herein mayutilize CDRS defined according to any of these systems, althoughpreferred embodiments use Kabat or Chothia defined CDRs. As used herein,“antibody variable domain” refers to the portions of the light and heavychains of antibody molecules that include amino acid sequences ofComplementarity Determining Regions (CDRs; ie., CDR1, CDR2, and CDR3),and Framework Regions (FRs). V_(H) refers to the variable domain of theheavy chain. V_(L) refers to the variable domain of the light chain.According to the methods used in this invention, the amino acidpositions assigned to CDRS and FRs may be defined according to Kabat(Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991)). Amino acid numbering ofantibodies or antigen binding fragments is also according to that ofKabat. CDRS can also be described as comprising amino acid residues froma “complementarity determining region” as defined by the IMGT, in someembodiments.

The term “multivalent binding protein” denotes a binding proteincomprising two or more antigen binding sites. A multivalent bindingprotein may be engineered to have three or more antigen binding sites,and is generally not a naturally occurring antibody. The term“multispecific binding protein” refers to a binding protein capable ofbinding two or more related or unrelated targets.

Similarily, unless indicated otherwise, the expression “multivalentantibody” is used throughout this specification to denote an antibodycomprising three or more antigen binding sites. For example, themultivalent antibody is engineered to have the three or more antigenbinding sites and is generally not a native sequence IgM or IgAantibody.

In some embodiments, the binding protein is a single chain dual variabledomain immunoglobulin protein. The terms “single chain dual variabledomain immunoglobulin protein” or “scDVD-Ig protein” or scFvDVD-Igprotein” refer to the antigen binding fragment of a DVD molecule that isanalogous to an antibody single chain Fv fragment. scDVD-Ig proteins aredescribed in U.S. Ser. Nos. 61/746,659; 14/141,498 (US application2014/0243228); and U.S. Ser. No. 14/141,500 (US application2014/0221621), which are incorporated herein by reference in theirentireties. In an embodiment, the variable domains of a scDVD-Ig proteinare antibody variable domains. In an embodiment, the variable domainsare non-immunoglobulin variable domains (e.g., receptor).

In some embodiments, the binding protein is a DVD-Fab. The terms“DVD-Fab” or fDVD-Ig protein” refer to the antigen binding fragment of aDVD-Ig molecule that is analogous to an antibody Fab fragment. fDVD-Igproteins are described in U.S. Ser. Nos. 61/746,663; 14/141,498 (USApplication 2014/0243228); and U.S. Ser. No. 14/141,501 (US applicationUS 2014/0235476), incorporated herein by reference in their entireties.

In some embodiments, the binding protein is a receptor DVD-Ig protein.The terms “receptor DVD-Ig protein” constructs, or “rDVD-Ig protein”refer to DVD-Ig™ constructs comprising at least one receptor-likebinding domain. rDVD-Ig proteins are described in U.S. Ser. Nos.61/746,616; and 14/141,499 (US application 2014/0219913), which areincorporated herein by reference in their entireties.

The term “receptor domain” (RD), or receptor binding domain refers tothe portion of a cell surface receptor, cytoplasmic receptor, nuclearreceptor, or soluble receptor that functions to bind one or morereceptor ligands or signaling molecules (e.g., toxins, hormones,neurotransmitters, cytokines, growth factors, or cell recognitionmolecules).

The terms multi-specific and multivalent IgG-like molecules or “pDVD-Ig”proteins are capable of binding two or more proteins (e.g., antigens).pDVD-Ig proteins are described in U.S. Ser. No. 14/141,502 (USApplication 2014/0213771), incorporated herein by reference in itsentirety. In certain embodiments, pDVD-Ig™ proteins are disclosed whichare generated by specifically modifying and adapting several concepts.These concepts include but are not limited to: (1) forming Fcheterodimer using CH3 “knobs-into-holes” design, (2) reducing lightchain missing pairing by using CH1/CL cross-over, and (3) pairing twoseparate half IgG molecules at protein production stage using “reductionthen oxidation” approach.

In certain embodiments, a binding protein disclosed herein is a“half-DVD-Ig” comprised of one DVD-Ig heavy chain and one DVD-Ig lightchain. The half-DVD-Ig™ protein preferably does not promotecross-linking observed with naturally occurring antibodies which canresult in antigen clustering and undesirable activities. See U.S. PatentPublication No. 2012/0201746 which is incorporated by reference hereinin its entirety. In some embodiments, the binding protein is a pDVD-Igprotein. In one embodiment, a pDVD-Ig construct may be created bycombining two halves of different DVD-Ig molecules, or a half DVD-Igprotein and half IgG molecule.

In some embodiments, the binding protein is an mDVD-Ig protein. As usedherein “monobody DVD-Ig protein” or “mDVD-Ig protein” refers to a classof binding molecules wherein one binding arm has been renderednon-functional. mDVD-Ig proteins are described in U.S. Ser. No.14/141,503 (US Application 2014/0221622) incorporated herein byreference in its entirety.

The Fc regions of the two polypeptide chains that have a formula ofVDH-(X1)n-C-(X2)n may each contain a mutation, wherein the mutations onthe two Fc regions enhance heterodimerization of the two polypeptidechains. In one aspect, knobs-into-holes mutations may be introduced intothese Fc regions to achieve heterodimerization of the Fc regions. SeeAtwell et al. (1997) J. Mol. Biol. 270:26-35.

In some embodiments, the binding protein is a cross-over DVD-Ig protein.As used herein “cross-over DVD-Ig” protein or “coDVD-Ig” protein refersto a DVD-Ig protein wherein the cross-over of variable domains is usedto resolve the issue of affinity loss in the inner antigen-bindingdomains of some DVD-Ig molecules. coDVD-Ig proteins are described inU.S. patent application Ser. No. 14/141,504, incorporated herein byreference in its entirety.

In certain embodiments, a binding protein that binds to DEspR (e.g., oneor any combination of human, cynomolgus, mouse and rat DEspR) isprovided as part of a bispecific antibody. The term “bispecificantibody”, as used herein, refers to full-length antibodies that aregenerated by quadroma technology (see Milstein et al. (1983) Nature 305:537-540), by chemical conjugation of two different monoclonal antibodies(see Staerz et al. (1985) Nature 314: 628-631), or by knob-into-hole orsimilar approaches which introduces mutations in the Fc region (seeHolliger et al. (1993) Proc. Natl. Acad. Sci. USA 90(14): 6444-6448),resulting in multiple different immunoglobulin species of which only oneis the functional bispecific antibody. By molecular function, abispecific antibody binds one antigen (or epitope) on one of its twobinding arms (one pair of HC/LC), and binds a different antigen (orepitope) on its second arm (a different pair of HC/LC). By thisdefinition, a bispecific antibody has two distinct antigen binding arms(in both specificity and CDR sequences), and is monovalent for eachantigen it binds.

The term “dual-specific antibody”, as used herein, refers to full-lengthantibodies that can bind two different antigens (or epitopes) in each ofits two binding arms (a pair of HC/LC) (see PCT Publication No. WO02/02773). Accordingly a dual-specific binding protein has two identicalantigen binding arms, with identical specificity and identical CDRsequences, and is bivalent for each antigen to which it binds.

A “functional antigen binding site” of a binding protein is one that iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In an exemplary embodiment, thedonor antibody is an antibody from a species different from the antibodyfrom which the framework regions are obtained or derived. In the contextof a humanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody providing or nucleic acid sequence encoding at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. In someembodiments, the term “acceptor” refers to the antibody amino acidproviding or nucleic acid sequence encoding the constant region(s). Inyet another embodiment, the term “acceptor” refers to the antibody aminoacid providing or nucleic acid sequence encoding one or more of theframework regions and the constant region(s). In a specific embodiment,the term “acceptor” refers to a human antibody amino acid or nucleicacid sequence that provides or encodes at least 80%, preferably, atleast 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues that does (do) not occur at one or more specific positions of ahuman antibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell known in the art, antibodies in development, or antibodiescommercially available).

Human heavy chain and light chain acceptor sequences are known in theart. In one embodiment of the disclosure the human heavy chain and lightchain acceptor sequences are selected from the sequences listed fromV-base (http://vbase.mrc-cpe.cam.ac.uk/) or from IMGT™ the internationalImMunoGeneTics Information System™.(http://imgt.cines.fr/textes/IMGTrepertoire/LocusGenes/). In anotherembodiment of the disclosure the human heavy chain and light chainacceptor sequences are selected from the sequences described in Table 3and Table 4 of U.S. Patent Publication No. 2011/0280800, incorporated byreference herein in their entireties.

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al. (2002) Crit. Rev. Immunol.22(3): 183-200; Marchalonis et al. (2001) Adv. Exp. Med. Biol.484:13-30). One of the advantages provided by various embodiments of thepresent disclosure stems from the recognition that germline antibodygenes are more likely than mature antibody genes to conserve essentialamino acid sequence structures characteristic of individuals in thespecies, hence less likely to be recognized as from a foreign sourcewhen used therapeutically in that species.

As used herein, the term “key” residues refer to certain residues withinthe variable domain that have more impact on the binding specificityand/or affinity of an antibody, in particular a humanized antibody. Akey residue includes, but is not limited to, one or more of thefollowing: a residue that is adjacent to a CDR, a potentialglycosylation site (can be either N- or O-glycosylation site), a rareresidue, a residue capable of interacting with the antigen, a residuecapable of interacting with a CDR, a canonical residue, a contactresidue between heavy chain variable domain and light chain variabledomain, a residue within the Vernier zone, and a residue in the regionthat overlaps between the Chothia definition of a variable heavy chainCDR/and the Kabat definition of the first heavy chain framework.

The term “Framework regions” (hereinafter “FR”) refers to the variabledomain residues that are not the CDR residues. Because the exactdefinition of a CDR sequence can be determined by different systems, themeaning of a framework sequence is subject to correspondingly differentinterpretations. The six CDRS (CDR-L1, -L2, and -L3 of light chain andCDR-H1, -H2, and -H3 of heavy chain) also divide the framework regionson the light chain and the heavy chain into four sub-regions (FR1, FR2,FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 andFR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Withoutspecifying the particular sub-regions as FR1, FR2, FR3 or FR4, aframework region, as referred by others, represents the combined FR'swithin the variable domain of a single, naturally occurringimmunoglobulin chain. As used herein, a FR represents one of the foursub-regions, and FRs represents two or more of the four sub-regionsconstituting a framework region. Without wishing to be bound by theory,each variable domain typically has four FRs identified as FR1, FR2, FR3and FR4. If the CDRS are defined according to Kabat, the light chain FRresidues are positioned at about residues 1-23 (LCFR1), 35-49 (LCFR2),57-88 (LCFR3), and 98-107 (LCFR4) and the heavy chain FR residues arepositioned about at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3),and 103-113 (HCFR4) in the heavy chain residues. If the CDRS compriseamino acid residues from hypervariable loops, the light chain FRresidues are positioned about at residues 1-25 (LCFR1), 33-49 (LCFR2),53-90 (LCFR3), and 97-107 (LCFR4) in the light chain and the heavy chainFR residues are positioned about at residues 1-25 (HCFR1), 33-52(HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain residues.In some instances, when the CDR comprises amino acids from both a CDR asdefined by Kabat and those of a hypervariable loop, the FR residues willbe adjusted accordingly. For example, when CDRH1 includes amino acidsH26-H35, the heavy chain FR1 residues are at positions 1-25 and the FR2residues are at positions 36-49.

An “isolated antibody” is intended to refer to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds DEspRis substantially free of antibodies that specifically bind antigensother than DEspR). An isolated antibody that specifically binds DEspRmay, however, have cross-reactivity to other antigens, such as DEspRmolecules from other species. In alternative embodiments, an isolatedantibody that specifically binds DEspR may specifically bind to thehuman DEspR molecule. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) and V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The term “linear antibodies” refers to the antibodies described inZapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)—C_(H)1-V_(H)—C_(H)1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The term “humanized antibody” refers to antibodies that comprise heavyand light chain variable domain sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. Accordingly, “humanized” antibodiesare a form of a chimeric antibody, that are engineered or designed tocomprise minimal sequence derived from non-human immunoglobulin. For themost part, humanized antibodies are human immunoglobulins (recipient oracceptor antibody) in which residues from a hypervariable region of therecipient are replaced by residues from a hypervariable region of anon-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies can comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992). As used herein, a “composite human antibody” or“deimmunized antibody” are specific types of engineered or humanizedantibodies designed to reduce or eliminate T cell epitopes from thevariable domains.

One type of humanized antibody is a CDR-grafted antibody, in which humanCDR sequences are introduced into non-human VH and VL sequences toreplace the corresponding nonhuman CDR sequences. Also “humanizedantibody” is an antibody or a variant, derivative, analog or fragmentthereof which immunospecifically binds to an antigen of interest andwhich comprises a framework (FR) region having substantially the aminoacid sequence of a human antibody and a complementary determining region(CDR) having substantially the amino acid sequence of a non-humanantibody. As used herein, the term “substantially” in the context of aCDR refers to a CDR having an amino acid sequence at least 80%, at least85%, at least 90%, at least 95%, at least 98% or at least 99% identicalto the amino acid sequence of a non-human antibody CDR. A humanizedantibody comprises substantially all of at least one, and typically two,variable domains (Fab, Fab′, F(ab′).sub.2, FabC, Fv) in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin (i.e., donor antibody) and all or substantially all ofthe framework regions are those of a human immunoglobulin consensussequence. In an embodiment, a humanized antibody also comprises at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. In some embodiments, a humanized antibody containsboth the light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain. A humanized antibody may beselected from any class of immunoglobulins, including IgM, IgG, IgD, IgAand IgE, and any isotype including without limitation IgG1, IgG2, IgG3,and IgG4. The humanized antibody may comprise sequences from more thanone class or isotype, and particular constant domains may be selected tooptimize desired effector functions using techniques well known in theart.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In an exemplary embodiment,such mutations, however, will not be extensive. Usually, at least 80%,preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (see, e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.If two amino acids occur equally frequently, either can be included inthe consensus sequence.

With respect to constructing DVD-Ig or other binding protein molecules,a “linker” is used to denote a single amino acid or a polypeptide(“linker polypeptide”) comprising two or more amino acid residues joinedby peptide bonds and used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see, e.g., Holligeret al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak (1994)Structure 2: 1121-1123).

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote et al. (1992) J. Mol. Biol., 224: 487-499, which isincorporated herein by reference. Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

A “human antibody,” “non-engineered human antibody,” or “fully humanantibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art. In one embodiment, the human antibody is selected froma phage library, where that phage library expresses human antibodies(Vaughan et al. Nature Biotechnology 14:309-314 (1996): Sheets et al.Proc. Natl. Acad. Sci. 95:6157-6162 (1998)); Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)). Human antibodies can also be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous mouse immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368:812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51(1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg andHuszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, the humanantibody can be prepared via immortalization of human B lymphocytesproducing an antibody directed against a target antigen (such Blymphocytes can be recovered from an individual or can have beenimmunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,147 (1):86-95 (1991); and U.S. Pat. No. 5,750,373.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result an improvement in the affinity ofthe antibody for antigen, compared to a parent antibody which does notpossess those alteration(s). Exemplary affinity matured antibodies willhave nanomolar or even picomolar affinities for the target antigen. Avariety of proceedures for producing affinity matured antibodies areknown in the art. For example, Marks et al. Bio/Technology 10:779-783(1992) describes affinity maturation by V_(H) and V_(L) domainshuffling. Random mutagenesis of CDR and/or framework residues isdescribed by: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813(1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995);and Hawkins et al., J. Mol. Biol. 226:889-896 (1992). Selective mutationat selective mutagenesis positions and at contact or hypermutationpositions with an activity enhancing amino acid residue is described inU.S. Pat. No. 6,914,128.

A “functional antigen binding site” of an antibody is one which iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same. For multimericantibodies, the number of functional antigen binding sites can beevaluated using ultracentrifugation analysis as described in Example 2of U.S. Patent Application Publication No. 20050186208. According tothis method of analysis, different ratios of target antigen tomultimeric antibody are combined and the average molecular weight of thecomplexes is calculated assuming differing numbers of functional bindingsites. These theoretical values are compared to the actual experimentalvalues obtained in order to evaluate the number of functional bindingsites.

As used herein, a “blocking” or “neutralizing” binding protein,antibody, antibody fragment, antigen-binding fragment or an antibody“antagonist” is one which inhibits or reduces the biological activity ofthe antigen it specifically binds to the antigen. For example, aDEspR-specific antagonist antibody, or binding protein binds DEspR andinhibits the ability of DEspR to, for example, bind VEGFsp and/oroptionally inhibits DEspR-induced angiogenesis, and can optionallyinhibit DEspR to induce vascular endothelial cell proliferation or toinduce vascular permeability. In certain embodiments, blocking orneutralizing antibodies or antagonist antibodies completely inhibit thebiological activity of the antigen. A neutralizing binding protein,antibody, antigen-binding fragment thereof as described herein can bindto DEspR resulting in the inhibition of a biological activity of theDEspR or other antigen. The neutralizing binding protein, antibody,antigen-binding fragment thereof can bind DEspR and reduce abiologically activity of the DEspR by at least about 20%, 40%, 60%, 80%,85%, or more. Inhibition of a biological activity of DEspR by aneutralizing binding protein, antibody or antigen-binding fragmentthereof can be assessed by measuring one or more indicators of DEspRbiological activity well known in the art; for example, inhibition ofDEspR to bind to VEGFsp-17 or VEGFsp-27 and/or optionally inhibitsDEspR-induced angiogenesis, or optionally inhibit DEspR to inducevascular endothelial cell proliferation or to induce vascularpermeability.

An antibody having a “biological characteristic” or “functionalcharacteristic”of a designated antibody is one which possesses one ormore of the biological properties of that antibody which distinguish itfrom other antibodies that bind to the same antigen, including, forexample, binding to a particular epitope, an EC50 value, IC50 value orK_(D)values, as defined elsewhere herein.

In order to screen for antibodies which bind to an epitope on an antigenbound by an antibody of interest, a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.

A “species-dependent antibody” is one which has a stronger bindingaffinity for an antigen from a first mammalian species than it has for ahomologue of that antigen from a second mammalian species. Normally, thespecies-dependent antibody “binds specifically” to a human antigen(i.e., has a binding affinity (K_(D)) value of no more than about 1X10⁻⁷M, preferably no more than about 1X10⁻⁸ M and most preferably no morethan about 1X10⁻⁹ M) but has a binding affinity for a homologue of theantigen from a second nonhuman mammalian species which is at least about50 fold, or at least about 500 fold, or at least about 1000 fold, weakerthan its binding affinity for the human antigen. The species-dependentantibody can be any of the various types of antibodies as defined above,but typically is a humanized or human antibody.

As used herein, “antibody mutant” or “antibody variant” refers to anamino acid sequence variant of the species-dependent antibody whereinone or more of the amino acid residues of the species-dependent antibodyhave been modified. Such mutants necessarily have less than 100%sequence identity or similarity with the species-dependent antibody. Inone embodiment, the antibody mutant will have an amino acid sequencehaving at least 75% amino acid sequence identity or similarity with theamino acid sequence of either the heavy or light chain variable domainof the species-dependent antibody, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%. Identity or similarity with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e., same residue) orsimilar (i.e., amino acid residue from the same group based on commonside-chain properties, see below) with the species-dependent antibodyresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence outside of the variable domain shall beconstrued as affecting sequence identity or similarity.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and can include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In certain embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined by,for example, the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, silver stain. Isolatedantibody includes the antibody in situ within recombinant cells since atleast one component of the antibody's natural environment will not bepresent. Ordinarily, however, isolated antibody will be prepared by atleast one purification step.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BlAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). See alsoJonsson U. et al., (1993) Ann. Biol. Clin., 51:19-26; Jonsson U. et al.,(1991) BioTechniques, 11:620-627 (1991); Johnsson U. et al., (1995) J.Mol. Recognit., 8:125-131; and Johnsson U. et al., (1991) Anal.Biochem., 198:268-277.

The term “binding protein conjugate” or “antibody conjugate” refers to abinding protein or antibody or antigen-binding fragment thereof asdescribed herein chemically linked to a second chemical moiety, such asa therapeutic or cytotoxic agent. The term “agent” is used herein todenote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materials.Preferably the therapeutic or cytotoxic agents include, but are notlimited to, anti-cancer therapies as discussed herein, as well aspertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. When employed in the contextof an immunoassay, a binding protein conjugate or antibody conjugate maybe a detectably labeled antibody, which is used as the detectionantibody.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (_(e.g. At)²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

The terms “crystal” and “crystallized” as used herein, refer to abinding protein, antibody or antigen-binding protein, or antigen bindingportion thereof, that exists in the form of a crystal. Crystals are oneform of the solid state of matter that is distinct from other forms suchas the amorphous solid state or the liquid crystalline state. Crystalsare composed of regular, repeating, three-dimensional arrays of atoms,ions, molecules (e.g., proteins such as DVD-Igs), or molecularassemblies (e.g., antigen/binding protein complexes).

By “fragment” is meant a portion of a polypeptide, such as a bindingprotein, antibody or antibody fragment, or antigen-binding portionthereof thereof, or nucleic acid molecule that contains, preferably, atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of theentire length of the reference nucleic acid molecule or polypeptide. Afragment can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200,300, 400, 500, 600, or more nucleotides or 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 120, 140, 160, 180, 190, 200 amino acids or more.

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide, a polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Itshould be understood that the anti-angiogenesis agent includes thoseagents that bind and block the angiogenic activity of the angiogenicfactor or its receptor. For example, an anti-angiogenesis agent is anantibody or other antagonist to an angiogenic agent as definedthroughout the specification or known in the art, e.g., but are notlimited to, antibodies to VEGF-A or to the VEGF-A receptor (e.g., KDRreceptor or Flt-1 receptor), VEGF-trap, anti-PDGFR inhibitors such asGLEEVEC™ (Imatinib Mesylate). Anti-angiogensis agents also includenative angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See,e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol., 53:217-39 (1991);Streit and Detmar, Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listinganti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo,Nature Medicine 5:1359-1364 (1999); Tonini et al., Oncogene,22:6549-6556 (2003) (e.g., Table 2 listing known antiangiogenicfactors); and Sato. Int. J. Clin. Oncol., 8:200-206 (2003) (e.g., Table1 lists anti-angiogenic agents used in clinical trials).

The term “anti-cancer therapy” refers to a therapy useful in treatingcancer. Examples of anti-cancer therapeutic agents include, but are notlimited to, e.g., surgery, chemotherapeutic agents, growth inhibitoryagents, cytotoxic agents, agents used in radiation therapy,anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, andother agents to treat cancer, such as anti-HER-2 antibodies (e.g.,HERCEPTIN®), anti-CD20 antibodies, an epidermal growth factor receptor(EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFRinhibitor (e.g., erlotinib (TARCEVA®)), platelet derived growth factorinhibitors (e.g., GLEEVEC™ (Imatinib Mesylate)), a COX-2 inhibitor(e.g., celecoxib), interferons, cytokines, antagonists (e.g.,neutralizing antibodies) that bind to one or more of the followingtargets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGFreceptor(s), TRAIL/Apo2, and other bioactive and organic chemicalagents, etc. Combinations thereof are also included in the invention.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents include, butare not limited to, alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaIl (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (TARCEVA®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above. Other chemotherapeutic agents that canbe used with compositions and methods described herein are disclosed inUS Publication No. 20080171040 or US Publication No. 20080305044 and areincorporated in their entirety by reference.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” as used herein refers to a compound orcomposition which inhibits growth of a cell in vitro and/or in vivo.Thus, the growth inhibitory agent can be one which significantly reducesthe percentage of cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), TAXOL®, and topo II inhibitors such as doxorubicin,epirubicin, daunorubicin, etoposide, and bleomycin. Those agents thatarrest G1 also spill over into S-phase arrest, for example, DNAalkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13.

The terms “antibody drug conjugate” or “antibody-drug conjugate,” asused herein, refer to an antibody conjugated to a non-proteinaceousagent, typically a chemotherapeutic agent, e.g., a cytotoxic agent, acytostatic agent, a toxin, or a radioactive agent. A linker molecule canbe used to conjugate the drug to the antibody. A wide variety of linkersand drugs useful in ADC technology are known in the art and can be usedin embodiments described herein. (See, for example, US20090028856;US2009/0274713; US2007/0031402; WO2005/084390; WO2009/099728; U.S. Pat.No. 5,208,020; U.S. Pat. No. 5,416,064; U.S. Pat. Nos. 5,475,092;5,585,499; 6,436,931; 6,372,738; and 6,340,701, all incorporated hereinby reference in their entireties). By combining the unique targeting ofmonoclonal antibodies or fragments thereof with the cancer-killingability of cytotoxic drugs, antibody drug conjugates allow sensitive andincreased discrimination between healthy and diseased tissue.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs described herein include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,.beta.-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

By “radiation therapy” is meant the use of directed gamma rays or betarays to induce sufficient damage to a cell so as to limit its ability tofunction normally or to destroy the cell altogether. It will beappreciated that there will be many ways known in the art to determinethe dosage and duration of treatment. Typical treatments are given as aone time administration and typical dosages range from 10 to 200 units(Grays) per day.

The term “intravenous infusion” refers to introduction of a drug intothe vein of an animal or human subject over a period of time greaterthan approximately 5 minutes, preferably between approximately 30 to 90minutes, although, according to the invention, intravenous infusion isalternatively administered for 10 hours or less. The term “intravenousbolus” or “intravenous push” refers to drug administration into a veinof an animal or human such that the body receives the drug inapproximately 15 minutes or less, preferably 5 minutes or less.

The term “subcutaneous administration” refers to introduction of a drugunder the skin of an animal or human subject, preferable within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle. The pocket can be created by pinchingor drawing the skin up and away from underlying tissue.

The term “subcutaneous infusion” refers to introduction of a drug underthe skin of an animal or human subject, preferably within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle for a period of time including, but notlimited to, 30 minutes or less, or 90 minutes or less. Optionally, theinfusion can be made by subcutaneous implantation of a drug deliverypump implanted under the skin of the animal or human subject, whereinthe pump delivers a predetermined amount of drug for a predeterminedperiod of time, such as 30 minutes, 90 minutes, or a time periodspanning the length of the treatment regimen.

The term “subcutaneous bolus” refers to drug administration beneath theskin of an animal or human subject, where bolus drug delivery ispreferably less than approximately 15 minutes, more preferably less than5 minutes, and most preferably less than 60 seconds. Administration ispreferably within a pocket between the skin and underlying tissue, wherethe pocket is created, for example, by pinching or drawing the skin upand away from underlying tissue.

A “disorder” is any condition that would benefit from treatment with,for example, an antibody described hereim. This includes chronic andacute disorders or diseases including those pathological conditionswhich predispose the mammal to the disorder in question. Non-limitingexamples of disorders to be treated herein include cancer; benign andmalignant tumors; leukemias and lymphoid malignancies; neuronal, glial,astrocytal, hypothalamic and other glandular, macrophagal, epithelial,stromal and blastocoelic disorders; and inflammatory, angiogenic andimmunologic disorders.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to thepolypeptide. The label can be itself be detectable (e.g., radioisotopelabels or fluorescent labels) or, in the case of an enzymatic label, cancatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.Preferably, the subject is a human. Patients are also subjects herein.

The terms “decrease,” “reduce,” “reduced”, “reduction”, “decrease,” and“inhibit” are all used herein generally to mean a decrease by astatistically significant amount relative to a reference. However, foravoidance of doubt, “reduce,” “reduction” or “decrease” or “inhibit”typically means a decrease by at least 10% as compared to a referencelevel and can include, for example, a decrease by at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, up to andincluding, for example, the complete absence of the given entity orparameter as compared to the reference level, or any decrease between10-99% as compared to the absence of a given treatment. Reduce orinhibit can refer to, for example, the symptoms of the disorder beingtreated, the presence or size of metastases or micrometastases, the sizeof the primary tumor, the presence or the size of the dormant tumor, orthe size or number of the blood vessels in angiogenic disorders.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) difference, above or below a reference value. Additionaldefinitions are provided in the text of individual sections below.

BRIEF DESCRIPTION OF THE FIGURES

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1C show representative brain images at acute onset of strokeusing a stroke-prone transgenic-hyperlipidemic, hypertensive rat model.FIG. 1A. Brain surface showing hemorrhages. FIG. 1B. H&E section confirmhemorrhages. FIG. 1C. H&E section show hemorrhagic infarct.

FIGS. 2A-2E show that a rat stroke model exhibits stroke-pathologylesions seen in humans. These lesions were detectable on ex vivo 11.7TMR-imaging using gradient echo sequences (FIG. 2A) and T2weighted MRI(FIGS. 2B-C). Ischemia surrounding microhemorrhages were noted onT2-weighted intensity analysis (FIG. 2C) and on analysis of T2relaxation time (FIGS. 2D-2E).

FIG. 3 demonstrates that survival analysis reveals significantshortening of life-span due to early stroke onset in transgenic females(black dots)>transgenic males (black open circles)/non-transgenicfemales (filled squares) >non-transgenic males (open squares).

FIG. 4 shows effect of anti-DEspR treatment on stroke survival in Tg25stroke-prone Dahl S rat model (Dahl S rats transgenic for humancholesteryl ester transfer protein). Tg25 female rats were treated (IVinfusion) with a single dose of either 10 μg of Isotype control (IgG1,n=10) or 10 μg of anti-DEspR 10A3H10 monoclonal antibody (mAb) (n=6) atstroke onset (rats were 4-6 months of age with documented neurologicaldeficits). Rats were allowed to proceed to recovery up to eventualdeath. As shown below, there is a significant increase in post-strokesurvival upon anti-DEspR treatment (Mean post-stroke survival time forcontrols=2.35±1.27 days versus Mean post-stroke survival time foranti-DEspR treated group=25.5±7.3 days; P=0.0002, Log-Rank Test)extending post-stroke survival>ten fold compared with controls.

FIG. 5A At stroke onset, a rat presented with dystonic head movements,which resolved completely after 3 days (FIG. 5B). Only 1 treatment wasgiven. The rat was then monitored until repeat stroke onset (thusdemonstrating stroke-prone phenotype).

FIG. 6A Masson-trichrome stained histology section showing tumor vesselswith loss of integrity of endothelial layer. Higher magnification showsloss of endothelial integrity and exposure of tumors cells to vascularlumen—a direct route for metastasis. FIG. 6B. After treatment withanti-rat DEspR monoclonal antibody (mAb) (10A3H10), tumors are smaller,and exhibit normalization of tumor blood vessels as seen inimmunohistochemistry staining (DAB) of DEspR expression in tumorendothelial cells and tumor cells.

FIG. 7 shows Western blot analysis of pulldown proteins from membraneproteins isolated from human glioblastoma tumor cells (U87), PNGase-Ftreated U87 pulldown proteins, and from hDEspR-Cos 1 permanenttransfectant cells.

FIGS. 8A-8B shows that two different monoclonal antibodies (mAbs) raisedagainst different domains in the DEspR protein bind to the identicalprotein bands on a Western blot of pull-down proteins (FIGS. 8A, 8B),thus refuting the existence of a stop codon at amino acid #14 positionand indicating DEspR protein in human cancer cells. FIG. 8A. Schematicdiagram of human DEspR (SEQ ID NO: 3). FIG. 8B. Western blot of pulldownproducts.

FIG. 9 shows Western blot (wb) analysis of 5G12E8 anti-DEspR monoclonalantibody (mAb) pulldown proteins: control non-digested (Control) andPNGase (PNG) digested pulldown proteins show loss of major 17 kDaprotein band and appearance of smaller-size bands indicatingdeglycosylation. WB probed with 5G12E8 mAb; 15 kDa molecular weight (mw)marker.

FIGS. 10A-10B show Western blot analysis of recombinant galectin-1protein probed with FIG. 10A anti-human galectin1 monoclonal antibody(mAb), and FIG. 10B anti-humanDEspR monoclonal antibody (mAb) 5G12E8.

FIG. 11 shows that concordant with pulldown of a DEspR-galectin1complex, DEspR and galectin1 colocalize in tumor cells at the invasivefront { } of a human glioblastoma (U87-csc) xenograft subQ tumor.Immunofluorescnce analysis of DEspR and galectin1; DAPI nuclear DNAstain; colocalized DEspR+galectin1. U87 glioblastoma CSC xenograft tumorin nude rats.

FIG. 12 shows invasive tumor cells (□) have invaded through thexenograft tumor's fibrous cap { }, through the overlying looseconnective tissue and aligned with a blood vessel in the tissuesurrounding the xenograft tumor cap. Representative photomicrograph of aglioblastoma U87-csc xenograft tumor (subcutaneous, 2 cm diameter)showing immunofluorescence detection of DEspR and galectin-1colocalization in invasive tumor cells. Using human-specific antibodiesfor both DEspR, Galectin-1, only human U87-tumor cells exhibitexpression. Co-localization (merged) is detected in invasive tumor cells(arrow) which are deduced to have invaded through the xenograft tumor'sfibrous cap { }, through the overlying loose connective tissue andaligned with a blood vessel in the tissue surrounding the xenografttumor cap. hDEspR, galectin1, DAPI, Merged [DEspR+gal1] +DIC(differential interference contrast).

FIG. 13 shows displacement of fluorescently labeled 7C5B2 binding toPanc1 CSCs by different peptide ligands. 50,000 cells were reacted with10 82 g/ml AF568-labelled 7C5B2 monoclonal antibody (mAb) at 4° C. for20 min in the absence or presence of the different peptides at 100×molar excess. Samples were immediately subjected to FACS analysis forquantification of DEspR+ cells. Samples were run in duplicates. AngII,angiotensinII; ET1, endothelin-1 (DEspR ligand); sp17, VEGFsp17 (DEspRligand); sp26, VEGFsp26 (DEspR ligand). Data presented as Mean±SD.

FIG. 14 shows testing of 5G12E8, 7C5B2 and 6G8G7 anti-hDEspR monoclonalantibodies (mAbs) on CSC-growth. 2,000 CSCs were seeded in 200 μL ofcomplete MammoCult media in an ultra-low attachment 96-well plate. Cellswere either not treated (control) or treated with mAbs (200 μg/ml) atdays 0, 2 and 4. Live cells were counted using Trypan Blue at day 5.Each experiment was run in five replicates. Data is presented asMean±SEM. ***P<0.001 (One Way ANOVA followed by Holm-Sidak MultipleComparisons Testing).

FIG. 15 shows comparative analysis of 7C5B2, 6G8G7, vh5/vk1 and vh3/vk2antibodies using the identical assay and following identicalexperimental conditions. Saturation binding curves of monoclonalantibody (mAb) binding to DEspR on Panc1 CSCs. Each mAb was labeled withAlexa Flour 568 using the Alexa Fluor 568 Monoclonal Antibody LabelingKit (Invitrogen). Specific binding was determined by FACS analysis using100,000 cells in 0.25 ml containing increasing concentrations of mAbs.Incubations were done at 4° C. for 20 min and immediately subjected toFACS analysis on a BDTM LSRII Flow cytometer. Each data point wasperformed in duplicate.

FIG. 16 shows in vitro inhibition of CSC growth by vh3/vk2 (vh3),vh5/vk1 (vh5), 6G8G7 and 7C5B2 mAbs. 2000 CSCs were seeded in 200 μL ofcomplete MammoCult media in an ultralow attachment 96-well plate inabsence (control) or presence (treated) of 100 μg/ml (7C5B2, 6G8G7), 300μg/ml (vh5/vk1) and 450 μg/ml (vh3/vk2) of corresponding monoclonalantibodies (mAbs). Cells were treated at days 0, 2 and 4. After 5 daysin culture live cells were counted using Trypan Blue. Each condition wasperformed in six replicates. Data is presented as Mean±SEM. ***, P<0.001(One-way ANOVA followed by Holm-Sidak test for multiple comparisons).

FIG. 17 shows inhibition of Panc1, MB231 and MB468 CSC-dependent in vivotumour initiation by 7C5B2 and vh5/vk1 monoclonal antibodies (mAbs).2×10⁶ CSCs in 200 μL of M2 media were incubated at 4° C. for 60 min inthe presence of 100 μg/ml of IgG2b (Isotype control), 100 μg/ml of 7C5B2mAb and 300 μg/ml of vh5/vk1 Ab and immediately injected subcutaneously(two injection sites per nude rat). Each condition was performed in 8-10replicates for CSCs subcutaneous tumour initiation. Subcutaneous tumourvolumes [Volume=4/3pi(a/2×b/2×c/2)] were measured 10 days after cellinjections. A significant inhibition of MB468, MB231, Panc1 CSCderivedsubcutaneous tumour formation was observed (7C5B2 55%, vh5 50% forMB468; 7C5B2 60%, vh5 48% for MB231; 7C5B2 72%, vh5 61% for Panc1). ***P<0.001 (One Way ANOVA followed by Holm-Sidak Test for multiplecomparisons).

FIG. 18 shows inhibition of U87 CSC-dependent tumour initiation andprogression in vivo by 5G12E8 and vh5/vk1 monoclonal antibodies (mAbs).3×10⁵ CSCs in 200 μL of M2 media were incubated at 4° C. for 60 min inthe absence (control) and presence of 300 μg/m1 of 5G12E8 and 900 μg/mlof vh5/vk1 mAb and immediately injected subcutaneously (two injectionsites per nude rat). Each condition was performed in 8 replicates forsubcutaneous tumour initiation. Tumour volume was calculated asVolume=4/3 pi (a/2×b/2×c/2).

FIGS. 19A-19C show testing efficacy in vivo of 5G12E8 and VH5/VK1pre-treatment of Panc1 and HCT116 CSCs on survival of nude ratsharbouring Panc1 and HCT116 peritoneal tumours. 2×10⁶ Panc1 (FIG. 19A,FIG. 19B) and HCT116 (FIG. 19C) CSCs in 200 μL of M2 media wereincubated at 4° C. for 60 min in the absence (control, in FIG. 19A, FIG.19B and FIG. 19C) and presence of 200 μg/ml of 5G12E8 (FIG. 19A) and 1mg/ml of VH5/VK1 Ab (FIG. 19B and FIG. 19C). Cells were immediatelyinjected intra-peritoneally. For FIG. 19A: controls n=8, 5G12E8-treatedn=8. For FIG. 19B: controls n=8, VH5/VK1-treated n=5; For FIG. 19C:controls n=2, VH5/VK1-treated n=2 (pilot).

FIGS. 20A-2B show anti-hDEspR monoclonal antibody (mAb) treatmentdecreases NSCLC (H460-csc) tumor progression resulting in increasedsurvival. H460 cancer stem-like cells (CSCs) xenograft tumors in nuderats (iv-infused). Post-cell injection (PCI)-in days. CSC injection ofH460-CSCs via tail vein: 100,000 CSCs mAb Tx: anti-hDEspR 6G8G7 mAbonset: 4 days PCI, 2× per week×4 weeks: 1 mg/kg body weight.

FIGS. 21A-21B show anti-hDEspR monoclonal antibody (mAb) decreases tumorprogression, and increases survival: pancreatic peritoneal metastasisnude rat model. mAb dose: 1 mg/kg ip; One Way ANOVA followed by HolmSidak Multiple Comparisons Test: p=0.002 (7C5B2 vs saline); p=0.0002(6g8g7 vs Saline); Gemcitabine (max dose: 26 mg/kg×4) vs saline: notsignificant.

FIG. 22 shows anti-hDEspR monoclonal antibody (mAb) decreases tumorinitiation/tumorigenesis in the pancreatic peritoneal metastasis nuderat model resulting in increased survival. 2,000,000 Panc1 CSCs in 200μL of M2 media were incubated at 4° C. for 60 min in the absence(control, Saline in FIG. 22) and presence of 200 μg/ml of 5G12E8 (FIGS.22) and 200 μg/ml of 6G8G7 mAb (FIG. 22). Cells were immediatelyinjected intra-peritoneally. 6G8G7 vs saline P=0.02; 5G12E8 vs salineP=0.03 (Log-Rank test followed by Holm-Sidak MCT).

FIG. 23 shows antiDEspR monoclonal antibodies (mAbs) (7C5B2, 6G8G7)decrease collagen-1 (col1) secretion by Panc1-CSCs. Panc1-CSC peritonealtumors co-express collagen-1 and DEspR. [human-specific col1/3 mAb;6G8G7]. Incubation 48 hrs at 37 ° C. (tissue culture incubator);Collagen measured by ELISA 6G8G7=200 μg/ml; 7C5B2=200 μg/ml. One WayANOVA+Holm-Sidak MCT; * P<10⁻³; ** P<10⁻⁷.

FIG. 24 shows Panc1-CSC s co-express collagen-1 and aSMA, a marker forEMT.

FIG. 25 shows anti-DEspR 6G8G7 decreases aSMA expression induced byTNF-α. αSMA expression is a marker of EMT; TNF-α is known to increasetumor growth and invasiveness of pancreatic cancer.

FIG. 26 shows detection of spliced DEspR RNA in human cancer cells(CSCs) by ARMS. ARMS assay specific for spliced DEspR-RNA 270-bpamplicon (arrow) spanning the spliced exon-to-exon junction confirmspresence of spliced DEspR-RNA in Panc1 (lane 1) and U87 (lane 2) CSCs.

FIG. 27 shows that Western blot analysis detects DEspR˜10 kDa protein inhuman cancer cells. Using 5G12E8 monoclonal antibody (mAb), twodifferent detection systems, two concentrations of mAb (1×, 2×) twoindependent Western blot analyses detected the predicted size for DEspRprotein in U87 CSC membrane protein isolates.

FIG. 28 shows Western Blot analysis of DEspR-galectin1, Rab1b,TMED10-complex. No antibody-cross reactivity observed. Anti-hDEspR5G12E8 monoclonal antibody (mAb) is a mouse monoclonal, hence secondary(2nd) Ab used for WB-detection reacts with mouse IgG1 backbone of5G12E8.

FIG. 29 shows immunohistofluorescence analysis of heterotopic (subQ)xenograft U87-csc tumors in nude rats. Detection ofhDEspR+/galectin+invading human tumor U87 cells along a blood vesseloutside the xenograft tumor capsule (below). Anti-hDEspR andanti-hGalectin1 detect human proteins only respectively.

FIG. 30 shows inhibition of angiogenesis by VEGFsp26. 10000 HUVECs wereseeded on P96 wells containing Matrigel. Cells were incubated overnightat 37° C. in M200 complete media in the absence (control) or presence ofVEGFsp26 (0.5-10 nM). Number of polygons, total tube length andbranching points was measured after 16 hours of incubation. Each datapoint was run in four replicates. Data is presented as mean percentagefrom control±SEM. VEGFsp26 IC50=1.185±0.1 nM based on number ofpolygons; VEGFsp26 IC₅₀=1.897±0.82 nM based on total tube length.

FIG. 31 shows stimulation of angiogenesis by VEGFsp17. 10000 HUVECs wereseeded on P96 wells containing Matrigel. Cells were incubated overnightat 37° C. in M200 media (without supplements) in the absence (control)or presence of VEGFsp17 (0.5-10 nM). Total tube length, number ofpolygons and number of branching points was measured after 16 hours ofincubation. Data is presented as Mean±SEM. Each data point was run in4-5 replicates. Curve fitting and EC50 values were obtained by using aSigmoidal dose-response model (GraphPad Prism 5.04). VEGFsp17EC₅₀=2.63±0.83 nM based on number of polygons; VEGFsp17 EC₅₀=1.98±1.19nM based on total tube length.

FIG. 32 shows inhibition of CSC-growth by VEGFsp26. 2000 CSCs wereseeded in 200 μL of complete MammoCult media in an ultra-low attachment96-well plate. Cells were either not treated (control) or treated withVEGFsp26 peptide (100 nM) at days 0, 2 and 4. Live cells were countedusing Trypan Blue at day 5. Data is presented as Mean±SEM. Eachexperiment was run in five replicates. *** P<0.001 (One Way ANOVAfollowed by Holm-Sidak MCT).

FIG. 33 shows inhibition of high fat diet-induced obesity by 10A3H10monoclonal antibody (mAb). Seven male Sprague Dawley rats were fed aHigh Fat Diet (HFD) starting at 9 weeks of age along with 4 male SpragueDawley rats maintained on a regular rat chow as controls (FIG. 33A,circle). After 6 weeks on the HFD (15 weeks of age, Week 0 on treatmentperiod) 3 HFD-rats were maintained on the HFD for the specified time ascontrols (FIG. 33A, circle) and 4 HFD-rats began treatment with anti-ratDEspR mAb (10A3H10): 50 μg/rat IV (T-F, 2×week) for 6 weeks (FIG. 33A,circle). Data is presented as Mean±SEM. For panel A *** P<0.001 (HFD vs10a3h10/reg diet), Two Way ANOVA followed by Student-Newman-Keuls testfor MC. For panels B-F, *** P<0.001; ** P<0.01; * P<0.05 (One Way ANOVAfollowed by Holm-Sidak test for MC).

FIG. 34 shows ELISA data from 6G8 monoclonal antibody (mAb) -derivedcandidate chimeric antibodies. Candidate 1 chimeric antibody wasselected for humanization based on ELISA testing of binding affinity tothe antigenic peptide: EMKSRWNWGS (SEQ ID NO: 2). Briefly, Candidate 1exhibited an EC50=1.87±0.6 ug/ml, which is a 2-fold improvement over the6g8 murine mAb with an EC50=4.96±1.4 ug/ml.

FIGS. 35A-35B show comparative binding affinity analysis. FIG. 35A.Binding of the top 2 6G8-IgG4 candidates shows the candidate with thebetter Bmax for binding to the antigenic peptide 6G8IgG4 or humab1),compared to 6g8IgG4κh3 or humab2. FIG. 35B. Binding affinity of theoriginal 6G8 mumab. It is noted that the secondary antibody detectionsystems are different as one is anti-human IgG4 for the candidate 6G8human monoclonal antibodies (mAbs), and the other is anti-mouse IgG2bfor the 6G8 mumab.

FIG. 36 shows studies of binding affinity to intact DEspR+ cells of6G8IgG4 humanized monoclonal antibody 1 (mAb 1) compared to 6G8-murinemonoclonal antibody (mAb).

FIG. 37 shows a representative fluorescence intensity plot. FACSanalysis of DEspR+ human cells was done using a red-fluorophore labeledanti-DEspR 6G8IgG4 humanized monoclonal antibody 1 (mAb 1) leadcandidate probing DEspR+ human cells. The peak is shifted to the rightindicating binding of fluorescently-labeled antibody to cells. Thelightest peak indicates the control isotype background.

FIG. 38 shows comparative analysis of 6G8 murine monoclonal antibody andlead candidate 6G8IgG4 humanized monoclonal antibody in inhibiting ratneutrophil survival. Neutrophils were isolated from stroke-prone ratsand incubated with the test and control antibodies at 37° C. for 4hours. Afterwards, live/dead cells were then counted and expressed as %of control.

FIGS. 39A-39D show testing of functional activity of a 6G8IgG4 humanizedmonoclonal antibody lead candidate for inhibition of angiogenesis invitro. FIG. 39A. Representative HUVECs bFGF-mediated, VEGF-independentangiogenesis in non-treated HUVECs control. FIG. 39B. Representativeimage of angiogenesis assay results with 6G8 humanized monoclonalantibody treatment of HUVECs. FIGS. 39C-3D. Marked inhibition ofangiogenesis complex network formation by 6G8 humanized monoclonalantibody lead candidate assessing polygons (FIG. 39C) and branch points(FIG. 39D).

FIGS. 40A-4 B show dose-dependent inhibition of HUVECs angiogenesis inbFGF-mediated/VEGF-independent angiogenesis assay. Measurement ofcomplex network formation was done using the following parameters: FIG.40A. % polygons compared to contemporaneous controls, FIG. 40B. % branchpoints compared to contemporaneous controls.

FIGS. 41A-41B demonstrate that a one-time treatment with an anti-ratDEspR 10A3 IgG1 antibody at 40 μg/kg/dose intravenously administered viatail vein injection prevents cerebral edema/microbleed progression atthe acute stroke stage. Survival analysis: Log Rank (Mantel-Cox) Test:p=0.0001; Control: n=10; Treated: n=7. Median survival for controlanimals was 0.5 days, while median survival for treated animals was 22days.

FIG. 42 shows immunofluorescence staining of brains tissue sections atstroke onset where increased DEspR expression compared to minimal to noexpression in normal age-matched brains. Immunostaining was done with a10A3 murine monoclonal antibody on fixed (PBS-buffered paraformaldehyde,pH 7.4), paraffin embedded 5 micron sections after antigen retrieval.Antibodies are labeled: Anti-DEspR: AF568; Anti-alpah smooth muscleactin: AF488 as + control.

FIGS. 43A-43B demonstrate that treatment with an anti-rat DEspR 10Aa3IgG1 antibody, at 1 mg/kg/dose intravenously administered via tail veininjection for 1 time per week for six weeks, delays stroke onset in thespTg25 rat model of spontaneous cerebral ischemic hemorrhagic-infarct.Survival analysis: Log Rank Test: p=0.0017; Control: n=5; Treated: n=6.Median survival for control animals was 13 days, while median survivalfor treated animals was 120 days.

FIGS. 44A-44B show PK evidence for in vivo efficacy. Plasmadose-concentration and target bioeffects of a prototype murinemonoclonal antibody in a stroke-prone rat model are shown.

FIGS. 45A-45B show PD evidence for in vivo efficacy. Braintarget-engagement and target-bioeffects of a prototype 6g8 murinemonoclonal antibody in a stroke-prone rat model are shown.

FIG. 46 shows that anti-DEspR monoclonal antibody-receptor interactionresults in internalization of fluorescently-labeled anti-human DEspR7C5-murine monoclonal antibody, transport to nucleus, and induction ofapoptosis. Representative time series of internalization offluorescently labeled (AF568) anti-DEspR monoclonal antibody by Panc1tumor cells within 1.5 hours is shown. Confocal images showingrepresentative Panc1 tumor cells from baseline (t-0) prior to additionof AF568-labeled antibody, up to 1 hour, 15 minutes from addition offluorescently labeled anti-DEspR 7C5 murine monoclonal antibody.Increasing intracellular fluorescence is detected in multiple Panc1cells.

FIG. 47 shows that anti-DEspR monoclonal antibody-receptor interactionresults in internalization of fluorescently-labeled anti-human DEspR7C5-monoclonal antibody, transport to nucleus and induction ofapoptosis. Representative time series of internalization offluorescently labeled (AF568) anti-DEspR monoclonal antibody by Panc1tumor cells within 1.5 hours at higher magnification of Panc1 tumorcells at baseline and t-75 minutes with corresponding brightfield imagesof Panc1 cells. At t-60 minutes, representative image of Panc1 tumorcells exposed to control AF568-labeled IgG2b isotype, with DAPI stainednuclei to mark cells, demonstrate no intracellular AF568 fluorescenceuptake.

FIG. 48 shows that internalization of anti-DEspR monoclonal antibody inPanc1 tumor cell is associated with nuclear changes consistent withearly phase of apoptosis. For comparison, see MORPHOLOGICAL ASPECTS OFAPOPTOSIS. Walter Malorni, Stefano Fais1, and Carla FiorentiniLaboratory of Ultrastructures and (1) Virology, Istituto Superiore diSanita',viale Regina Elena 299, 00161, Rome, Italy.

FIG. 49 shows that internalization of anti-DEspR monoclonal antibody inPanc1 tumor cell is associated with nuclear changes consistent withearly phase of apoptosis. For comparison, see Indian Journal of Cancer,Vol. 50, No. 3, July-September, 2013, pp. 274-283, “Various methodsavailable for detection of apoptotic cells.”

FIG. 50 shows internalization of anti-human DEspR 7C5-humanizedmonoclonal antibody (VH5NK1) by DEspR+ Panc1-CSCS. These data regardingstable interaction betweem anti-DEspR monoclonal antibody and DEspRsupport their efficacy in inhibiting cancer cells, as well as intargeting of cancer cells and cancer stem-like cells for delivery oftherapeutics or for in vivo detection of DEspR+ cancer cells and cancerstem-like cells. Notably, internalization was observed with bothanti-humanDEspR murine monoclonal antibody and humanized monoclonalantibody.

FIG. 51 demonstrates that FACS analyses detect DEspR expression in CSCsin multiple cancers and that multiple anti-human DEspR monoclonalantibodies targeting 2 different DEspR epitopes, namely 5G12E8 and 7C5B2for epitope 1, and 6G8 for epitope 2, inhibit CSC growth.

FIG. 52 demonstrates a comparison of a prototype 6G8 murine monoclonalantibody with 6G8 humanized monoclonal antibodies: binding to DEspR onintact DEspR+ cells (Panc1 tumor cells). Binding of AF-568-labeledmonoclonal antibodies to Panc1 cells at 4° C. for 20 minutes wasanalyzed. Binding was quantified by FACS with corresponding isotypelabeled antibodies as background controls. These data demonstratehigh-affinity binding of 6G8 humanized monoclonal antibodies to DEspR onintact cells having a native target, not just the antigenic peptide.Furthermore, 6G8 humanized monoclonal antibodies were developed usingtwo different human IgG Fc regions—human IgG1 and hinge-stabilized humanIgG4, thus affirming high-affinity binding.

FIG. 53 shows a comparison of functional activities of 6G8 humanizedmonoclonal antibodies and 6G murine monoclonal antibody on inhibition ofrat neutrophil survival. Neutrophil survival assays were performed withfreshly isolated rat neutrophils. Neutrophils (50,000/well) wereincubated in the absence or presence of monoclonal antibodies at 37° C.for 4 hrs and live cells counted by using Trypan blue. Each poinnt wasdone in triplicates.

FIG. 54 shows a comparison of functional activities of 6G8 humanizedmonoclonal antibodies and 6G murine monoclonal antibody on inhibition ofHUVECs angiogenesis. HUVEC-tube formation (angiogenesis) assay (20,000cells/well) was performed in the presence or absence of monoclonalantibodies. Number of polygons, number of branch points (standardparameters of complex network formation) were determined after 14 hrs ofincubation at 37° C. ND, not determined.

DETAILED DESCRIPTION

Provided herein are compositions comprising novel anti-DEspR antibodiesand DEspR-binding fragments thereof derived from the 6G8G7 and 7C5B2anti-DEspR antibodies, and methods of their use in anti-angiogenesis andanti-tumor proliferation and invasiveness therapies, such as thetreatment of cancer, as well as the treatment of diseases wherepathological angiogenesis plays a role, such as in carotid arterydisease, vasa vasorum neovascularization, and stroke, and vulnerableplaque neovascularization and consequent heart disease.

As described herein, peptide GSNEMKSRWNWGS (SEQ ID NO: 1) was used as anantigenic peptide to generate monoclonal antibodies specific for DEspR.A smaller or partial peptide EMKSRWNWGS (SEQ ID NO: 2) was then used toscreen for monoclonal antibodies and narrow down the potential epitopefor the antibodies generated using the peptide of SEQ ID NO: 1, such asthe monoclonal antibody and derivatives thereof termed herein as “6G8G7”or “6G8” or “6g8g7” or “6g8.” Such antibodies or antigen-bindingfragments thereof or derivatives thereof can also be referred to as an“antibody or antigen-binding fragment specific for and/or directed toSEQ ID NO: 1 or SEQ ID NO: 2.” The inventors have discovered that the6G8G7 anti-DEspR antibody is a neutralizing antibody that inhibitsmultiple mechanisms contributing to tumor recurrence in vitro anddecreases in vivo tumor initation and progression significantly, thusincreasing survival using a pancreatic peritoneal metastasis nude ratmodel. In addition, the data provided herein demonstrate that the 6G8G7anti-DEspR antibody and derivatives thereof decreases tumorinitiation/tumorigenesis of Panc1-CSCs, decreases collagen-1 (col1)secretion by Panc1-CSCs, and decreases alpha smooth muscle acitn (αSMA)expression induced by the major inflammatory cytokine, TNF-α.

Despite these and other data, DEspR is still annotated as a “pseudogene”in the NCBI database. Accordingly, the compositions comprisingantibodies and antigen-binding fragments thereof that bind to DEspRdescribed herein, and the epitopes used to generate these antibodies andantigen-binding fragments, provide novel and unexpected results fortreating cancer and other disorders dependent on aberrant angiogenesis,and on DEspR-roles in tumor initiation, recurrence, therapy resistance,microvessel leakiness, microbleeds. Thus, provided herein arecompositions and methods comprising isolated antibodies andantigen-binding fragments having one or more functional characteristicsand anti-DEspR antibodies and antigen-binding fragments thereof derivedfrom the 6G8G7 and 7C5B2 anti-DEspR antibodies, including chimeric andhumanized antibodies, for use in treatment of angiogenesis-dependentdiseases or disorders.

Anti-DEspR Antibodies and Antigen-Binding Fragments Thereof

The dual endothelin-1/VEGF signal peptide activated receptor (DEspR),also known as DEAR, was originally cloned from a Dahl salt-sensitivehypertensive rat brain cDNA library and was shown to be a singletransmembrane receptor coupled to a Ca2+-mobilizing transduction pathwaybinding endothelin-1 (ET-1) and angiotensin-II (Ang II) with equivalentaffinities (Ruiz-Opazo N. et al. (1998), Molecular characterization of adual Endothelin-1/Angiotensin II Receptor. Mol Med. 4: 96-108).Subsequent molecular studies elucidated that the mouse ortholog does notinteract with AngII but binds ET-1 and the vascular endothelial growthfactor signal peptide (VEGFsp) with equal affinities instead.

The role of DEspR in cancer has been deduced, in part, from itsembryonic-lethal null mutation phenotype resulting in E10.5-12.5 dayembryonic lethality characterized by abnormal vasculogenesis withincomplete dorsal aorta formation, and by absent angiogenesis, andfailed endocardial-to-mesenchymal transition/migration resulting inthin-walled hearts. The DEspR null mouse phenotype is similar to, but isdistinguished from the heterozygous VEGF+/− knockout mouse phenotype,and from the homozygous knockout mouse phenotype of its overlappingopposite-strand transcript, Fbxw7, a ubiquitin ligase oncosuppressor bythe detection of hyperconvoluted neuroepithelium throughout the neuraltube, indicating a DEspR-specific role in neuroepithelial stemcell-to-radial cell transition and/or migration. Furthermore, 50%reduction of DEspR expression in heterozygous DEspR+/− knockout mice isnot embryonic lethal, in contrast to the embryonic-lethal phenotype ofheterozygous VEGF+/− knockout mice, and decreased tumor growth inDEspR+/− male mice, in polar contrast to increased tumorigenesisexpected from the loss of Fbxw7-tumor suppressor functions as seen inhuman cancer and mouse tumor inactivating mutations. Importantly, DEspRinhibition at the protein level via an anti-ratDEspR-specific polyclonalantibody decreased tumor growth, tumor vascularization, and nuclearmalignancy-grade in irradiation-induced rat mammary tumors, therebyclarifying DEspR-specific pro-tumorigenic roles in contrast to the tumorsuppressor roles of Fbxw7. Confirmatory immunohistochemistry detectedDEspR+ expression not only in rat mammary tumor blood vessels, but alsoin tumor cells and in invading tumor cells. Additionally,DEspR-signaling, studied in human DEspR-positive permanent Cos1-celltransfectants phosphorylates Akt in a dose-response manner. “DEspR rolesin tumor vasculo-angiogenesis, invasiveness, CSC-survival and anoikisresistance: a ‘common receptor coordinator’ paradigm,” Herrera V L etal., PLoS One. 2014 Jan 21;9(1):e85821.

More recent studies show that DEspR is a common receptor expressed intumor cells, microvessels, and anchorage-independent cancer stem cells(CSCs), with differential expression in cell-and nuclear-membranes, aswell as in the cytoplasm. DEspR is differentially increased in bothhuman pancreatic cancer and glioblastoma in contrast to adjacent normaltissue. DEspR-inhibition at the protein level decreased in vitroangiogenesis, tumor cell invasiveness, CSC-cell anoikis resistance,survival, and promoted pro-apoptosis balance. Concordantly,DEspR-inhibition also decreased in vivo Panc1- and U87-CSC-xenografttumor volumes, vasculogenesis, invasiveness, and tumor cell survival inthe expanding tumor zone.

However, despite these data, DEspR is still annotated as a “pseudogene”in the NCBI database. Accordingly, the compositions comprisingantibodies and antigen-binding fragments thereof that bind to DEspRdescribed herein, and the epitopes used to generate these antibodies andantigen-binding fragments, provide novel and unexpected results fortreating cancer and other disorders dependent on aberrant angiogenesis.

The term “ DEspR,” as used herein, refers to the 85-amino acid dualendothelin/VEGF signal peptide receptor (DEspR) having the human aminoacid native sequence of:MTMFKGSNEMKSRWNWGSITCIICFTCVGSQLSMSSSKASNFSGPLQLYQRELEIFIVLTDVPNYRLIKENSHLHTTIVDQGRTV (SEQ ID NO: 3), as described by, e.g., AccessionNumber EF212178.1, Gene ID 102191832, or Glorioso et al. 2007, togetherwith naturally occurring allelic, splice variants, and processed formsthereof. Typically, as used herein, DEspR refers to human DEspR of SEQID NO: 3.The term “DEspR” is also used to refer to truncated forms orfragments of the polypeptide comprising specific amino acids sequencesof the 85-amino acid human dual endothelin/VEGF signal peptide receptor.Reference to any such forms of DEspR can be identified in theapplication, e.g., by “DEspR (1-9)” or amino acids 1-9 of SEQ ID NO: 3.

As used herein a DEspR “native sequence” or DEspR “wild-type sequence”polypeptide comprises a polypeptide having the same amino acid sequenceas a DEspR polypeptide derived from nature. Thus, a native sequencepolypeptide can have the amino acid sequence of naturally-occurringpolypeptide from any mammal. Such native sequence polypeptide can beisolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence” polypeptide specifically encompassesnaturally-occurring truncated or secreted forms of the polypeptide(e.g., an extracellular domain sequence), naturally-occurring variantforms (e.g., alternatively spliced forms) and naturally-occurringallelic variants of the polypeptide.

As used herein, a DEspR polypeptide “variant” means a biologicallyactive DEspR polypeptide having at least about 80% amino acid sequenceidentity with a native sequence of a DEspR polypeptide. Such variantsinclude, for instance, polypeptides wherein one or more amino acidresidues are added, or deleted, at the N- or C-terminus of thepolypeptide. Ordinarily, a variant has at least about 80% amino acidsequence identity, more preferably at least about 90% amino acidsequence identity, and even more preferably at least about 95% aminoacid sequence identity with the native sequence polypeptide.

DEspR is a cell-membrane receptor that binds to endothelin-1, to VEGFsignal peptide signal peptide 26 (VEGFsp-26/sp26, FIG. 13), and to VEGFsignal peptide 17 (VEGFsp-17/sp17, FIG. 13). VEGFsp-26 has the humansequence MNFLLSWVHWSLALLLYLHHAKWSQA (SEQ ID NO: 4). VEGFsp-17 has thehuman sequence MNFLLSWVHWSLALLLY (SEQ ID NO: 48).

Provided herein are compositions and methods comprising antibodies andantigen-binding fargements thereof, such as anti-DEspR antibodies orantigen-binding fragments thereof that are capable of neutralizing,blocking, inhibiting, abrogating, reducing, or interfering with DEspRactivities including its binding to endothelin-1 or VEGFsp.

Accordingly, in some aspects, provided herein are anti-DEspR antibodiesor antibody fragments thereof that specifically bind human DEspR of SEQID NO: 3 and reduce or inhibit DEspR biological activity.

In some aspects, provided herein are anti-DEspR antibodies orantigen-binding fragments thereof specific for an epitope of DEspRcomprising, consisting essentially of, or consisting of GSNEMKSRWNWGS(SEQ ID NO: 1).

In some aspects, provided herein are anti-DEspR antibodies orantigen-binding fragments thereof specific for an epitope of DEspRcomprising, consisting essentially of, or consisting of EMKSRWNWGS (SEQID NO: 2).

In some embodiments of this aspect and all such aspects describedherein, the anti-DEspR antibody or antigen-binding fragment thereof thatbinds to DEspR and inhibits DEspR biological activity blocks or inhibitsinteraction of DEspR with VEGFsp comprising the sequence of SEQ ID NO:4.

In some aspects, provided herein are anti-DEspR antibodies orantigen-binding fragments thereof that bind the same or an overlappingepitope of DEspR (e.g., an epitope of human DEspR) as any one of theantibodies produced by hybridomas 6G8G7 and 7C5B2. In some embodimentsof this aspect, the anti-DEspR antibodies or antigen-binding fragmentsthereof bind the same or overlapping epitope of SEQ ID NO: 1 or SEQ IDNO: 2. As known to one of ordinary skill in the art, antibodies thatrecognize and bind to the same or overlapping epitopes of DEspR (e.g.,human DEspR) can be identified using routine techniques such as animmunoassay, for example, by showing the ability of one antibody toblock the binding of another antibody to a target antigen, i.e. , acompetitive binding assay. Competition binding assays also can be usedto determine whether two antibodies have similar binding specificity foran epitope. Competitive binding can be determined in an assay in whichthe immunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as DEspR. Numerous types ofcompetitive binding assays are known, for example: solid phase direct orindirect radioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli C et al.,(1983) Methods Enzymol 9: 242-253); solid phase direct biotin-avidin EIA(see Kirkland T N et al., (1986) J Immunol 137: 3614-9); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor Press); solid phase direct label RIA using 1-125 label (see MorelG A et al., (1988) Mol Immunol 25(1): 7-15); solid phase directbiotin-avidin EIA (Cheung R C et al., (1990) Virology 176: 546-52); anddirect labeled RIA. (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82). Typically, such an assay involves the use of purified antigen(e.g., DEspR) bound to a solid surface or cells bearing either of these,an unlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition can be measured by determining the amount oflabel bound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least50-55%, 55-60%, 60-65%, 65-70% 70-75% or more. A competition bindingassay can be configured in a large number of different formats usingeither labeled antigen or labeled antibody. In a common version of thisassay, the antigen is immobilized on a 96-well plate. The ability ofunlabeled antibodies to block the binding of labeled antibodies to theantigen is then measured using radioactive or enzyme labels. For furtherdetails see, for example, Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68: 269-274;Kuroki M et al., (1990) Cancer Res 50: 4872-4879; Kuroki Metal., (1992)Immunol Invest 21: 523-538; Kuroki Metal., (1992) Hybridoma 11: 391-407and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D editors supra,pp. 386-389. A competition assay can be performed, for example, usingsurface plasmon resonance (BIACORE) e.g., by an ‘in tandem approach’such as that described by Abdiche Y N et al., (2009) Analytical Biochem386: 172-180, whereby DEspR antigen is immobilized on the chip surface,for example, a CMS sensor chip and the anti-DEspRantibodies are then runover the chip. To determine if an antibody competes with a givenanti-DEspR antibody or antigen-binding fragment thereof describedherein, the anti-DEspR antibody is first run over the chip surface toachieve saturation and then the potential, competing antibody is added.Binding of the competing antibody can then be determined and quantifiedrelative to a non-competing control.

Competition binding assays can be used to determine whether an antibodyis competitively blocked, e.g., in a dose dependent manner, by anotherantibody for example, an antibody that binds essentially the sameepitope, or overlapping epitopes, as a reference antibody, when the twoantibodies recognize identical or sterically overlapping epitopes incompetition binding assays such as competition ELISA assays, which canbe configured in all number of different formats, using either labeledantigen or labeled antibody.

Accordingly, in some of the aspects described herein, an antibody orantigen-bdining fragment can be generated comprising any of thesequences described herein, including any of the one or more V_(H) CDRSand/or one or more V_(L) CDRs of any one of the antibodies produced byhybridomas 6G8G7 and 7C5B2 as described herein, and variants thereof.

Accordingly, in some embodiments of the aspects described herein, ananti-DEspR antibody can be tested in competition binding assays with anyone of the antibodies produced by hybridomas 6G8G7 and 7C5B2 describedherein, or a chimeric or Fab antibody thereof, or an anti-DEspR antibodycomprising one or more V_(H) CDRS and one or more V_(L) CDRs of any oneof the antibodies produced by hybridomas 6G8G7 and 7C5B2 describedherein.

Affinities of anti-DEspR antibodies and antigen-binding fragmentsthereof can be determined, for example, by a surface plasmon resonancebased assay (such as the BIACORE assay described in PCT ApplicationPublication No. WO2005/012359); enzyme-linked immunoabsorbent assay(ELISA); and competition assays (e.g. RIA's), for example. An anti-DEspRantibody for use in the compositions and methods described herein can besubjected to other biological activity assays, e.g., in order toevaluate its effectiveness as a therapeutic, or its effectiveness as adiagnostic aid, etc. Such assays are known in the art and depend on thetarget antigen and intended use for the antibody. Examples include theHUVEC inhibition assay; tumor cell growth inhibition assays (asdescribed in WO 89/06692, for example); antibody-dependent cellularcytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays(U.S. Pat. No. 5,500,362); and agonistic activity or hematopoiesisassays (see WO 95/27062). Other biological activity assays that can beused to assess an anti-DEspR antibody are described herein in theExamples section and in “DEspR roles in tumor vasculo-angiogenesis,invasiveness, CSC-survival and anoikis resistance: a ‘common receptorcoordinator’ paradigm,” Herrera V L et al., PLoS One. 2014 Jan21;9(1):e85821, the contents of which are herein incorporated byreference in their entireties. Thus, anti-DEspR antibodies or antibodyfragments thereof that are useful in the compositions and methodsdescribed herein include any antibodies or antibody fragments thereofthat bind with sufficient affinity and specificity to DEspR, i.e., arespecific for DEspR, and can reduce or inhibit the biological activity ofDEspR.

While it is preferred that the anti-DEspR antibodies and antigen-bindingfragments thereof used in the compositions and methods described hereinare monoclonal, in some embodiments, polyclonal antibodies can first beraised or generated in animals by multiple subcutaneous (sc) orintraperitoneal (ip) injections of the relevant antigen, e.g., SEQ IDNO: 1 or SEQ ID NO: 2 and an adjuvant. It can be useful, in someembodiments, to conjugate the relevant antigen to a protein that isimmunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹are different alkyl groups. Methods of generating such polyclonalantibodies to a given antigenic sequence, such as SEQ ID NO: 1 or SEQ IDNO:2, are known to one of ordinary skill in the art.

Preferably, anti-DEspR antibodies or antibody fragments thereof for usewith the compositions and methods described herein are anti-DEspRmonoclonal antibodies or fragments thereof.

In some aspects, the anti-DEspR monoclonal antibody is a monoclonalanti-DEspR antibody 6G8G7 produced or expressed by the hybridoma 6G8G7described herein, and referred to as a “6G8G7 variant antibody” or“6G8G7 anti-DEspR variant antibody” and derivatives or antigen-bindingfragments thereof, including, for example, a “6G8G7 variant variableheavy chain,” or a “6G8G7 variant variable light chain.” In someaspects, the anti-DEspR monoclonal antibody is a monoclonal anti-DEspRantibody 7C5B2 produced or expressed by the hybridoma 7C5B2 and referredto as a “variant 7C5B2” or “variant 7C5B2 anti-DEspR antibody” andderivatives or antigen-binding fragments thereof, including, forexample, a “7C5B2 variant variable heavy chain,” or a “7C5B2 variantvariable light chain.”

As described herein, the 6G8G7 and 7C5B2 hybridomas produce monoclonalantibodies, termed herein as a “6G8G7 variant antibody” or “6G8G7variant” or a “variant 7C5B2 anti-DEspR antibody” or a “variant 7C5B2antibody” that is highly specific for DEspR and can potently inhibitDEspR biological activity. The biological characteristics of 6G8G7 and7C5B2 anti-DEspR variant antibodies, and any chimeric or humanizedantibodies or antigen-binding fragments derived or generated therefrom,render them particularly useful for the compositions and methodsdescribed herein, including therapeutic and diagnostic applications.

Accordingly, sequence analysis of 6G8G7 hybridoma was performed, asdescribed herein, to identify heavy and light chain variable domainsequences, and complementarity determining region (CDR) sequences, ofthe antibodies produced by the 6G8G7 hybridoma, and also to identifyheavy and light chain variable domain sequences and complementaritydetermining region (CDR) sequences of the antibodies produced by the7C5B2 hybridoma for use in the compositions and methods describedherein.

The nucleotide sequence encoding a V_(H) or variable domain of the heavychain of a 6G8G7 HV1 variant antibody, as obtained by sequence analysisof sequences obtained from a 6G8G7 hybridoma, is:

(SEQ ID NO: 5) GGATCCCAAGTGCAGCTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACCGGCTATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAATGGCTGGGAATGATTTGGGATGATGGAAGCACAGACTATAATTCAGCTCTCAAATCCAGACTGATCATCACCAAGGACAACTCCAGGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGGTACTACTGTGCCAGAGACCCAGTATAGGTCCATTTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATGGTGGCG GTGGTTCT

The amino acid sequence of the VH domain of a 6G8G7 HV1 variant antibodycorresponding to SEQ ID NO: 5 is:

(SEQ ID NO: 6) GSQVQLQESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWDDGSTDYNSALKSRLIITKDNSRSQVFLKMNSLQTDDTARYYCARDPVVHFYAMDYWGQGTSVTVSSAKTTPPSVYGGGGS

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(H) domain of SEQ ID NO: 6 of the 6G8G7 HV1 variantantibody according to the Kabat sequence numbering is: GYGVN (SEQ ID NO:7). The amino acid sequence of the CDR2 of the V_(H) domain of SEQ IDNO: 6 of the 6G8G7 HV1 variant antibody according to the Kabat sequencenumbering is: MIWDDGSTDYNSALKS (SEQ ID NO: 8). The amino acid sequenceof the CDR3 of the V_(H) domain of SEQ ID NO: 6 of the 6G8G7 HV1 variantantibody according to the Kabat sequence numbering is: DPVVHFYAMDY (SEQID NO: 9).

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(H) domain of SEQ ID NO: 6 of the 6G8G7 HV1 variantantibody according to the IMGT sequence numbering is: GFSLTGYG (SEQ IDNO: 10). The amino acid sequence of the CDR2 of the V_(H) domain of SEQID NO: 6 of the 6G8G7 HV1 variant antibody according to the IMGTsequence numbering is: IWDDGST (SEQ ID NO: 11).

The nucleotide sequence encoding a V_(H) or variable domain of the heavychain of a 6G8G7 HV2 variant antibody, as obtained by sequence analysisof sequences obtained from a 6G8G7 hybridoma, is:

(SEQ ID NO: 12) GGATCCGAAGTTCAGCTGCAGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGACTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTACGATAAACTATACGCCATCTCTAAAGGATAAATTCATCATTTCTAGAGACACCGCCAAAAAAACTCTGTACCTGCAAATGAGCAAAGTGAGATCAGAGGACACAGCCCTTTATTACTGTGCAAGACATGGTAGAGGTATGGACTACTGGAGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATGGTGGCGGTGGTTCT 

The amino acid sequence of the V_(H) domain of a 6G8G7 HV2 variantantibody corresponding to SEQ ID NO: 12 is:

(SEQ ID NO: 13) GSEVQLQESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDTAKKTLYLQMSKVRSEDTALYYCARHGRGMDYWSQGTSVTVSSAKTTPPSVYGGGGS

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(H) domain of SEQ ID NO: 13 of the 6G8G7 HV2 variantantibody according to the Kabat sequence numbering is: RYWMS (SEQ ID NO:14). The amino acid sequence of the CDR2 of the V_(H) domain of SEQ IDNO: 13 of the 6G8G7HV2 variant antibody according to the Kabat sequencenumbering is: EINPDSSTINYTPSLKD (SEQ ID NO: 15). The amino acid sequenceof the CDR3 of the V_(H) domain of SEQ ID NO: 13 of the 6G8G7HV2 variantantibody according to the Kabat sequence numbering is: HGRGMDY (SEQ IDNO: 16).

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(H) domain of SEQ ID NO: 13 of the 6G8G7 HV2 variantantibody according to the IMGT sequence numbering is: GFDFSRYW (SEQ IDNO: 17). The amino acid sequence of the CDR2 of the V_(H) domain of SEQID NO: 13 of the 6G8G7 HV2 variant antibody according to the IMGTsequence numbering is: INPDSSTI (SEQ ID NO: 18).

The nucleotide sequence encoding a V_(H) or variable domain of the heavychain of the 7C5B2 HV2 variant antibody, as obtained by sequenceanalysis of sequences obtained from a variant 7C5B2 hybridoma, is:

(SEQ ID NO: 19) CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACTGTCTCAGGATTCTCATTAAAAAGTTATGCTGTAAGCTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATCTGGGGTGACGGGAGCACAGATTATCATTCAGCTCTCATATCCAGACTGAGCATCAGTAAGGATAACTCCAAGAGCCAATTTTTCTTAAGACTGAACAGTCTGCAAACTGATGACACAGCCACGTATTACTGTGCCAGAGGAACTGGGACGGGGTTTGCTTACTGGGGCCAGGGGACTCTGGTCACTGTCTCTGCA

The amino acid sequence of the VH domain of the 7C5B2 HV2 variantantibody corresponding to SEQ ID NO: 19 is:

(SEQ ID NO: 20) QVQLKESGPGLVAPSQSLSITCTVSGFSLKSYAVSWVRQPPGKGLEWLGVIWGDGSTDYHSALISRLSISKDNSKSQFFLRLNSLQTDDTATYYCARGTG TGFAYWGQGTLVTVSA

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(H) domain of SEQ ID NO: 20 of the 7C5B2 HV2 variantantibody according to the Kabat sequence numbering is: SYAVS (SEQ ID NO:21). The amino acid sequence of the CDR2 of the V_(H) domain of SEQ IDNO: 20 of the 7C5B2 HV2 variant antibody according to the Kabat sequencenumbering is: VIWGDGSTDYHSALIS (SEQ ID NO: 22). The amino acid sequenceof the CDR3 of the V_(H) domain of SEQ ID NO: 20 of the 7C5B2 HV2variant antibody according to the Kabat sequence numbering is: GTGTGFAY(SEQ ID NO: 23).

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(H) domain of SEQ ID NO: 20 of the 7C5B2 HV2 variantantibody according to the IMGT sequence numbering is: GFSLKSYA (SEQ IDNO: 24). The amino acid sequence of the CDR2 of the V_(H) domain of SEQID NO: 20 of the 7C5B2 HV2 variant antibody according to the IMGTsequence numbering is: IWGDGSTD (SEQ ID NO: 25).

The nucleotide sequence encoding a V_(L) or variable domain of the lightchain of a 6G8G7 KV1 variant antibody, as obtained by sequence analysisof sequences obtained from a 6G8G7 hybridoma, is:

(SEQ ID NO: 26) GGTGGCGGTGGTTCTGATATTGTGCTCACACAAACTAACCAAATCATGTCCGCATCAGTAGGAGACCGGGTCAGTGTCACCTGCAAGGCCAGTCAGAATGTGGATAGTAATGTGGCCTGGTATCAACAGAAACCTGGACATTCTCCCAAAGCACTAATTTATTCGGCATCCTACCGGTACAGTAGAGTCCCTGATCGCATCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACCAATGTGCAGTCTAAAGACTTGGCAGACTATTTCTGTCAGCAATATCACAGCTATCCTCTTCTCGCGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCCTCGAG

The amino acid sequence of the V_(L) domain of a 6G8G7 KV1 variantantibody corresponding to SEQ ID NO: 26 is:

(SEQ ID NO: 27) GGGGSDIVLTQTNQIMSASVGDRVSVTCKASQNVDSNVAWYQQKPGHSPKALIYSASYRYSRVPDRITGSGSGTDFTLTITNVQSKDLADYFCQQYHSYPLLAFGAGTKLELKRADAAPTVSLE

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(L) domain of SEQ ID NO: 27 of the 6G8G7 KV1 variantantibody according to the Kabat sequence numbering is: KASQNVDSNVA (SEQID NO: 28). The amino acid sequence of the CDR2 of the V_(L) domain ofSEQ ID NO: 27 of the 6G8G7 KV1 variant antibody according to the Kabatsequence numbering is: SASYRYS (SEQ ID NO: 29). The amino acid sequenceof the CDR3 of the V_(L) domain of SEQ ID NO: 27 of the 6G8G7 KV1variant antibody according to the Kabat sequence numbering is: QQYHSYP(SEQ ID NO: 30).

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(L) domain of SEQ ID NO: 27 of the 6G8G7 KV1 variantantibody according to the IMGT sequence numbering is: QNVDSN (SEQ ID NO:31). The amino acid sequence of the CDR2 of the V_(L) domain of SEQ IDNO: 27 of the 6G8G7 KV1 variant antibody according to the IMGT sequencenumbering is: SAS (SEQ ID NO: 32).

The nucleotide sequence encoding a V_(L) or variable domain of the lightchain of a 6G8G7 KV8 variant antibody, as obtained by sequence analysisof sequences obtained from a 6G8G7 hybridoma, is:

(SEQ ID NO: 33) GGTGGCGGTGGTTCTGACATTGTGATCACACAGTCTAACGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAAGTTTCATGCACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGTAGTTACCCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACC AACTGTATCCCTCGAG

The amino acid sequence of the V_(L) domain of a 6G8G7 KV8 variantantibody corresponding to SEQ ID NO: 33 is:

(SEQ ID NO: 34) GGGGSDIVITQSNAIMSASPGEKVTITCSASSSVSFMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPLTFGAGTKLELKRADAAPTVSLE

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(L) domain of SEQ ID NO: 34 of the 6G8G7 KV8 variantantibody according to the Kabat sequence numbering is: SASSSVSFMH (SEQID NO: 35). The amino acid sequence of the CDR2 of the V_(L) domain ofSEQ ID NO: 34 of the 6G8G7 KV8 variant antibody according to the Kabatsequence numbering is: STSNLAS (SEQ ID NO: 36). The amino acid sequenceof the CDR3 of the V_(L) domain of SEQ ID NO: 34 of the 6G8G7 KV8variant antibody according to the Kabat sequence numbering is: QQRSSYP(SEQ ID NO: 37).

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(L) domain of SEQ ID NO: 33 of the 6G8G7 KV8 variantantibody according to the IMGT sequence numbering is: SSVSF (SEQ ID NO:38). The amino acid sequence of the CDR2 of the V_(L) domain of SEQ IDNO: 34 of the 6G8G7 KV8 variant antibody according to the IMGT sequencenumbering is: STS (SEQ ID NO: 39).

The nucleotide sequence encoding a V_(L) or variable domain of the lightchain of a 6G8G7 KV2 variant antibody, as obtained by sequence analysisof sequences obtained from a 6G8G7 hybridoma, is:

(SEQ ID NO: 40) GGTGGCGGTGGTTCTGATATTGTGCTCACACAGACTCACAAATTCCTGCTTGTATCAGCAGGAGACAGGATTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACTATGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGATGACCTGGCAGTTTATTTCTGTCAACAGGATTATAGCTCCCCGTTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGC ACCAACTGTATCCCTCGAG

The amino acid sequence of the V_(L) domain of a 6G8G7 KV2 variantantibody corresponding to SEQ ID NO: 40 is:

(SEQ ID NO: 41) GGGSDIVLTQTHKFLLVSAGDRITITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQADDLAVYFCQQDYSSPFTFGGGTKLEIKRADAAPTVSLE

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(L) domain of SEQ ID NO: 41 of the 6G8G7 KV2 variantantibody according to the Kabat sequence numbering is: KASQSVSNDVA (SEQID NO: 42). The amino acid sequence of the CDR2 of the V_(L) domain ofSEQ ID NO: 41 of the 6G8G7 KV2 variant antibody according to the Kabatsequence numbering is: YASNRYT (SEQ ID NO: 43). The amino acid sequenceof the CDR3 of the V_(L) domain of SEQ ID NO: 41 of the 6G8G7 KV2variant antibody according to the Kabat sequence numbering is: QQDYSSPFT(SEQ ID NO: 44).

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(L) domain of SEQ ID NO: 41 of the 6G8G7 KV2 variantantibody according to the IMGT sequence numbering is: QSVSND (SEQ ID NO:45). The amino acid sequence of the CDR2 of the V_(L) domain of SEQ IDNO: 41 of the 6G8G7 KV2 variant antibody according to the IMGT sequencenumbering is: YAS (SEQ ID NO: 46). The amino acid sequence of the CDR3of the V_(L) domain of SEQ ID NO: 41 of the 6G8G7 KV2 variant antibodyaccording to the IMGT sequence numbering is: QQDYSSP (SEQ ID NO: 47).

The nucleotide sequence encoding a V_(L) or variable domain of the lightchain of a 7C5B2 KV2 variant antibody, as obtained by sequence analysisof sequences obtained from a 7C5B2 hybridoma, is:

(SEQ ID NO: 49) GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCCGAGTGGAGGCTGAGGATCTGGGAATTTATTTCTGCTCTCAATGTACACATATTCCGTGGACGTTCGGTGGAGGCACCAACCTGGAAATCAAA

The amino acid sequence of the V_(L) domain of a 7C5B2 KV2 variantantibody corresponding to SEQ ID NO: 49 is:

(SEQ ID NO: 50) DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQCTHIP WTFGGGTNLEIK

The amino acid sequence of the complementarity determining region 1 orCDR1 of the V_(L) domain of SEQ ID NO: 50 of the 7C5B2 KV2 variantantibody according to the Kabat sequence numbering is: RSSQSLVHSNGNTYLH(SEQ ID NO: 51). The amino acid sequence of the CDR2 of the V_(L) domainof SEQ ID NO: 50 of the 7C5B2 KV2 variant antibody according to theKabat sequence numbering is: KVSNRFS (SEQ ID NO: 52). The amino acidsequence of the CDR3 of the V_(L) domain of SEQ ID NO: 50 of the 7C5B2KV2 variant antibody according to the Kabat sequence numbering is:SQCTHIPWT (SEQ ID NO: 53).

Accordingly, in some embodiments of the aspects provided herein, theheavy and/or light chain variable domain(s) sequence(s) of the different6G8G7 or 7C5B2 variant antibodies, i.e., SEQ ID NO: 6, SEQ ID NO: 13,SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 41, and/or SEQID NO: 50 can be used to generate, for example, chimeric, humanized, orcomposite human antibodies, as described elsewhere herein.

In some aspects, monoclonal antibodies that specifically bind to DEspRare provided having one or more biological characteristics of a 6G8G7 or7C5B2 variant antibody. As used herein, an antibody having a “biologicalcharacteristic” of a designated antibody, such as a 6G8G7 variantantibody, is one that possesses one or more of the biologicalcharacteristics of that antibody which distinguish it from otherantibodies that bind to the same antigen. For example, biologicalcharacteristics of the 6G8G7 monoclonal antibody includes having an ED₅₀value (i.e., the dose therapeutically effective in 50% of thepopulation) at or around the ED₅₀ value of the 6G8G7 antibody for thegiven population; having an EC₅₀ value (i.e., the dose that achieves ahalf-maximal inhibition of a given parameter or phenotype) at or aroundthe EC₅₀ value of the 6G8G7 antibody for a given parameter or phenotye.The effects of any particular dosage can be monitored by a suitablebioassay. For example, in some embodiments of these aspects, the givenparameter or phenotype to be inhibited by the antibody that specificallybinds to DEspR and has one or more biological characteristics of a 6G8G7or 7C5B2 variant antibody can include, but is not limited to, the meantotal tube number in an in vitro tubulogenesis assay, the mean totaltube length in an in vitro tubulogenesis assay, the mean number ofbranching points in an in vitro tubulogenesis assay, the mean number ofvessel connections in an in vitro tubulogenesis assay, and/or tumor cellinvasiveness.

In some embodiments of the aspects described herein, anti-DEspRantibodies for use in the compositions and methods described hereininclude monoclonal antibodies that bind to the same epitope or epitopesof DEspR as a monoclonal anti-DEspR 6G8G7 or 7C5B2 variant antibody. Insome embodiments of the aspects described herein, the epitope comprises,consists essentially of, or consists of SEQ ID NO: 1. In someembodiments of the aspects described herein, the epitope comprises,consists essentially of, or consists of SEQ ID NO: 2.

The DNA sequences encoding the antibodies or antibody fragment thatspecifically bind DEspR described herein can also be modified, forexample, by substituting the coding sequence for human heavy- andlight-chain constant domains or framework regions in place of thehomologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al.,Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide, as also described elsewhereherein.

Such non-immunoglobulin polypeptides can be substituted for the constantdomains of an antibody, or they can be substituted for the variabledomains of one antigen-combining site of an antibody to create achimeric bivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

Also provided herein, in some aspects, are humanized anti-DEspRantibodies for use in the compositions and methods described herein.Humanized forms of non-human (e.g., murine) antibodies refer to chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some embodiments, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies can compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, a humanized antibody can comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also can comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

A humanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) where substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies. In some embodiments, humanized antibodiescomprising one or more variable domains comprising the amino acidsequence of the variable heavy (SEQ ID NO: 20) and/or variable light(SEQ ID NO: 49) chain domains of the murine anti-DEspR antibody 7C5B2,are provided, for example.

Accordingly, in some embodiments of the aspects described herein, one ormore heavy and/or one or more light chain CDR regions of a humanizedanti-DEspR binding protein, binding protein, antibody or antibodyfragment, or antigen-binding portion thereof, or antigen-binding portionthereof thereof comprises a sequence of any of the 6G8G7 or 7C5B2variant antibodies described herein. In some such embodiments, the oneor more variable heavy chain CDR regions comprises a sequence selectedfrom the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO:22, and SEQ ID NO: 23. In some such embodiments, the one or morevariable light chain CDR regions comprises a sequence selected from thegroup consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43, SEQID NO: 44, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53. In some suchembodiments, the one or more variable heavy chain CDR regions comprisesa sequence selected from the group consisting of SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, and the one or more variablelight chain CDR regions comprises a sequence selected from the groupconsisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43, SEQ IDNO: 44, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53.

Accordingly, in some embodiments of the aspects provided herein, theheavy and/or light chain variable domain(s) sequence(s) of any of the6G8G7 or 7C5B2 variant antibodies, and their respective variable heavychain CDR regions SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQID NO: 23, and/or variable light chain CDR regions SEQ ID NO: 28, SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 52,and SEQ ID NO: 53, can be used to generate, for example, CDR-grafted,chimeric, humanized, or composite human antibodies or antigen-bindingfragments, as described elsewhere herein. As understood by one ofordinary skill in the art, any variant, CDR-grafted, chimeric,humanized, or composite antibodies or antigen-binding fragments derivedfrom any of the 6G8G7 or 7C5B2 variant antibodies or any one of theantibodies produced by the 6G8G7 or 7C5B2 hybridomas useful in thecompositions and methods described herein will maintain the ability toimmunospecifically bind DEspR, such that the variant, CDR-grafted,chimeric, humanized, or composite antibody or antigen-binding fragmentthereof has at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95% , at least 100%, or any amount greater than thebinding affinity to DEspR relative to the original antibody from whichit is derived.

In some such embodiments of the aspects described herein, the anti-DEspRantibody or antigen-binding fragment thereof comprises one, two, threeor four of the framework regions of a light chain variable regionsequence which is at least 75%, 80%, 85%, 90%, 95%, or 100% identical toone, two, three or four of the framework regions of the light chainvariable region sequence of the 6G8G7 or 7C5B2 variant antibodies fromwhich it is derived. In some embodiments of the aspects describedherein, the light chain variable framework region that is derived fromsaid amino acid sequence consists of said amino acid sequence but forthe presence of up to 10 amino acid substitutions, deletions, and/orinsertions, preferably up to 10 amino acid substitutions. In someembodiments of the aspects described herein, the light chain variableframework region that is derived from said amino acid sequence consistsof said amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 aminoacid residues being substituted for an amino acid found in an analogousposition in a corresponding non-human, primate, or human light chainvariable framework region. In some embodiments of the aspects describedherein, the antibody or antigen-binding fragment further comprises one,two, three or all four V_(L) framework regions derived from the V_(L) ofa human or primate antibody. The primate or human light chain frameworkregion of the antibody selected for use with the light chain CDRsequences described herein, can have, for example, at least 70% identitywith a light chain framework region of the non-human parent antibody.The primate or human antibody selected can have the same orsubstantially the same number of amino acids in its light chaincomplementarity determining regions to that of the light chaincomplementarity determining regions of any of the 6G8G7 or 7C5B2 variantantibodies. In some embodiments of the aspects described herein, theprimate or human light chain framework region amino acid residues arefrom a natural primate or human antibody light chain framework regionhaving at least 75% identity, at least 80% identity, at least 85%identity (or more) with the light chain framework regions of any of the6G8G7 or 7C5B2 variant antibodies. In some embodiments, the anti-DEspRantibody or antigen-binding fragment further comprises one, two, threeor all four V_(L) framework regions derived from a human light chainvariable kappa subfamily. In some embodiments, the anti-DEspR antibodyor antigen-binding fragment further comprises one, two, three or allfour VL framework regions derived from a human light chain variablelambda subfamily.

In some embodiments of the aspects described herein, the anti-DEspRantibody or antigen-binding fragment thereof comprises one, two, threeor all four of the framework regions of the heavy chain variable regionsequence of any of the 6G8G7 or 7C5B2 variant antibodies. In someembodiments of the aspects described herein, the anti-DEspR antibody orantigen-binding fragment thereof comprises one, two, three, or four ofthe framework regions of a heavy chain variable region sequence which isat least 75%, 80%, 85%, 90%, 95% or 100% identical to one, two, three orfour of the framework regions of the heavy chain variable regionsequence of any of the 6G8G7 or 7C5B2 variant antibodies. In someembodiments of the aspects described herein, the heavy chain variableframework region that is derived from said amino acid sequence consistsof said amino acid sequence but for the presence of up to 10 amino acidsubstitutions, deletions, and/or insertions, preferably up to 10 aminoacid substitutions. In some embodiments of the aspects described herein,the heavy chain variable framework region that is derived from saidamino acid sequence consists of said amino acid sequence with 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 amino acid residues being substituted for anamino acid found in an analogous position in a corresponding non-human,primate, or human heavy chain variable framework region. In someembodiments of the aspects described herein, the anti-DEspR antibody orantigen-binding fragment further comprises one, two, three or all fourV_(H) framework regions derived from the V_(H) of a human or primateantibody. The primate or human heavy chain framework region of theantibody selected for use with the heavy chain CDR sequences describedherein, can have, for example, at least 70% identity with a heavy chainframework region of the non-human parent antibody. Preferably, theprimate or human antibody selected can have the same or substantiallythe same number of amino acids in its heavy chain complementaritydetermining regions to that of the light chain complementaritydetermining regions of any of the 6G8G7 or 7C5B2 variant antibodies. Insome embodiments of the aspects described herein, the primate or humanheavy chain framework region amino acid residues are from a naturalprimate or human antibody heavy chain framework region having at least75% identity, at least 80% identity, at least 85% identity (or more)with the heavy chain framework regions of any of the 6G8G7 or 7C5B2variant antibodies. In specific embodiments, the antibody orantigen-binding fragment further comprises one, two, three or all fourV_(H) framework regions derived from a human heavy chain variablesubfamily (e.g., one of subfamilies 1 to 7).

In some embodiments of the aspects described herein, the position of oneor more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g.,CDR1, CDR2, or CDR3) region of an antibody described herein can vary,i.e., be shorter or longer, by one, two, three, four, five, or six aminoacid positions so long as immunospecific binding to DEspR (e.g., humanDEspR) is maintained (e.g., substantially maintained, for example, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% of the binding of the original antibody from which it isderived). For example, in some embodiments, the position defining a CDRof any of the 6G8G7 or 7C5B2 variant antibodies can vary, i.e., beshorter or longer, by shifting the N-terminal and/or C-terminal boundaryof the CDR by one, two, three, four, five, or six amino acids, relativeto the CDR position of any one of the antibodies described herein, solong as immunospecific binding to DEspR (e.g., human DEspR) ismaintained (e.g., substantially maintained, for example, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95% ofthe binding of the original antibody from which it is derived). Inanother embodiment, the length of one or more CDRs along the V_(H)(e.g., CDR1, CDR2, or CDR3) and/or V_(L) (e.g., CDR1, CDR2, or CDR3)region of an antibody described herein can vary (e.g., be shorter orlonger) by one, two, three, four, five, or more amino acids, so long asimmunospecific binding to DEspR (e.g., human DEspR) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95% of the binding ofthe original antibody from which it is derived).

With respect to the heavy chain, in some embodiments of the aspectsdescribed herein, the heavy chain of an antibody described herein can bean alpha (α), delta (α), epsilon (ε), gamma (γ) or mu (μ) heavy chain.In some embodiments of the aspects described herein, the heavy chain ofan antibody described can comprise a human alpha (α), delta (Δ), epsilon(ε), gamma (γ) or mu (μ) heavy chain. Non-limiting examples of humanconstant region sequences have been described in the art, e.g., see U.S.Pat. No. 5,693,780 and Kabat E A et al., (1991) supra.

In some embodiments of the aspects described herein, one, two or moremutations (e.g., amino acid substitutions) are introduced into the Fcregion of an any of the 6G8G7 or 7C5B2 variant antibodies describedherein or a fragment thereof (e.g., CH2 domain (residues 231-340 ofhuman IgG1) and/or CH3 domain (residues 341-447 of human IgG1) and/orthe hinge region, with numbering according to the Kabat numbering system(e.g., the EU index in Kabat)) to alter one or more functionalproperties of the antibody, such as serum half-life, complementfixation, Fc receptor binding and/or antigen-dependent cellularcytotoxicity.

In some embodiments of the aspects described herein, one, two or moremutations (e.g., amino acid substitutions) are introduced into the hingeregion of the Fc region (CH1 domain) such that the number of cysteineresidues in the hinge region are altered (e.g., increased or decreased)as described in, e.g., U.S. Pat. No. 5,677,425. The number of cysteineresidues in the hinge region of the CH1 domain can be altered to, e.g.,facilitate assembly of the light and heavy chains, or to alter (e.g.,increase or decrease) the stability of the antibody.

In some embodiments of the aspects described herein, one, two or moremutations (e.g., amino acid substitutions) are introduced into the Fcregion of an anti-DEspR antibody described herein or an antigen-bindingfragment thereof (e.g., CH2 domain (residues 231-340 of human IgG1)and/or CH3 domain (residues 341-447 of human IgG1) and/or the hingeregion, with numbering according to the Kabat numbering system (e.g.,the EU index in Kabat)) to increase or decrease the affinity of theantibody for an Fc receptor (e.g., an activated Fc receptor) on thesurface of an effector cell. Mutations in the Fc region of an antibodyor fragment thereof that decrease or increase the affinity of anantibody for an Fc receptor and techniques for introducing suchmutations into the Fc receptor or fragment thereof are known to one ofskill in the art. Examples of mutations in the Fc receptor of anantibody that can be made to alter the affinity of the antibody for anFc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos.WO 02/060919; WO 98/23289; and WO 97/34631, which are incorporatedherein by reference.

In some embodiments of the aspects described herein, one, two or moreamino acid mutations (i.e., substitutions, insertions or deletions) areintroduced into an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to alter (e.g., decreaseor increase) half-life of the antibody in vivo. See, e.g., InternationalPublication Nos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S.Pat. Nos. 5,869,046, 6,121,022, 6,277,375 and 6,165,745 for examples ofmutations that will alter (e.g., decrease or increase) the half-life ofan antibody in vivo.

In some embodiments of the aspects described herein, one, two or moreamino acid mutations (i.e., substitutions, insertions or deletions) areintroduced into an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to decrease the half-lifeof the anti-DEspR antibody in vivo. In some embodiments of the aspectsdescribed herein, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to increase the half-life of the antibody invivo. In some embodiments of the aspects described herein, theantibodies can have one or more amino acid mutations (e.g.,substitutions) in the second constant (CH2) domain (residues 231-340 ofhuman IgG1) and/or the third constant (CH3) domain (residues 341-447 ofhuman IgG1), with numbering according to the EU index in Kabat (Kabat EA et al., (1991) supra). In some embodiments of the aspects describedherein, the constant region of the IgG1 of an antibody orantigen-binding fragment thereof described herein comprises a methionine(M) to tyrosine (Y) substitution in position 252, a serine (S) tothreonine (T) substitution in position 254, and a threonine (T) toglutamic acid (E) substitution in position 256, numbered according tothe EU index as in Kabat. See U.S. Pat. No. 7,658,921, which isincorporated herein by reference. This type of mutant IgG, referred toas “YTE mutant” has been shown to display fourfold increased half-lifeas compared to wild-type versions of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281: 23514-24). In some embodiments of theaspects described herein, an antibody or antigen-binding fragmentthereof comprises an IgG constant domain comprising one, two, three ormore amino acid substitutions of amino acid residues at positions251-257, 285-290, 308-314, 385-389, and 428-436, numbered according tothe EU index as in Kabat.

In some embodiments of the aspects described herein, one, two or moreamino acid substitutions are introduced into an IgG constant domain Fcregion to alter the effector function(s) of the anti-DEspR antibody. Forexample, one or more amino acids selected from amino acid residues 234,235, 236, 237, 297, 318, 320 and 322, numbered according to the EU indexas in Kabat, can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments ofthe aspects described herein, the deletion or inactivation (throughpoint mutations or other means) of a constant region domain can reduceFc receptor binding of the circulating antibody thereby increasing tumorlocalization. See, e.g., U.S. Pat. Nos. 5,585,097 and 8,591,886 for adescription of mutations that delete or inactivate the constant domainand thereby increase tumor localization. In some embodiments of theaspects described herein, one or more amino acid substitutions may beintroduced into the Fc region of an antibody described herein to removepotential glycosylation sites on Fc region, which may reduce Fc receptorbinding (see, e.g., Shields R L et al., (2001) J Biol Chem 276:6591-604). In some embodiments of the aspects described herein, one ormore of the following mutations in the constant region of an antibodydescribed herein can be made: an N297A substitution; an N297Qsubstitution; a L235A substitution and a L237A substitution; a L234Asubstitution and a L235A substitution; a E233P substitution; a L234Vsubstitution; a L235A substitution; a C236 deletion; a P238Asubstitution; a D265A substitution; a A327Q substitution; or a P329Asubstitution, numbered according to the EU index as in Kabat. In someembodiments of the aspects described herein, an antibody orantigen-binding fragment thereof described herein comprises the constantdomain of an IgG1 with an N297Q or N297A amino acid substitution.

In some embodiments of the aspects described herein, one or more aminoacids selected from amino acid residues 329, 331 and 322 in the constantregion of an anti-In some embodiments of the aspects described herein,one or more amino acids selected from amino acid residues 329, 331 and322 in the constant region of an anti-DEspR antibody described herein,numbered according to the EU index as in Kabat, can be replaced with adifferent amino acid residue such that the antibody has altered C1qbinding and/or reduced or abolished complement dependent cytotoxicity(CDC). This approach is described in further detail in U.S. Pat. No.6,194,551 (Idusogie et al). In some embodiments of the aspects describedherein, one or more amino acid residues within amino acid positions 231to 238 in the N-terminal region of the CH2 domain of an antibodydescribed herein are altered to thereby alter the ability of theantibody to fix complement. This approach is described further inInternational Publication No. WO 94/29351. In some embodiments of theaspects described herein, the Fc region of an antibody described hereinis modified to increase the ability of the antibody to mediate antibodydependent cellular cytotoxicity (ADCC) and/or to increase the affinityof the antibody for an Fcγ receptor by mutating one or more amino acids(e.g., introducing amino acid substitutions) at the following positions:238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270,272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296,298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329,330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388,389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439, numberedaccording to the EU index as in Kabat. This approach is describedfurther in International Publication No. WO 00/42072.

In some embodiments of the aspects described herein, an anti-DEspRantibody described herein comprises the constant region of an IgG4antibody and the serine at amino acid residue 228 of the heavy chain,numbered according to the EU index as in Kabat, is substituted forproline.

Antibodies with reduced fucose content have been reported to have anincreased affinity for Fc receptors, such as, e.g., FcγRIIIa.Accordingly, in certain embodiments, the anti-DEspR antibodies orantigen-binding fragments thereof described herein have reduced fucosecontent or no fucose content. Such antibodies can be produced usingtechniques known to one skilled in the art. For example, the antibodiescan be expressed in cells deficient or lacking the ability offucosylation. In a specific example, cell lines with a knockout of bothalleles of α1,6-fucosyltransferase can be used to produce antibodieswith reduced fucose content. The POTELLIGENTR™ system (Lonza) is anexample of such a system that can be used to produce antibodies withreduced fucose content. Alternatively, antibodies or antigen-bindingfragments with reduced fucose content or no fucose content can beproduced by, e.g.: (i) culturing cells under conditions which prevent orreduce fucosylation; (ii) posttranslational removal of fucose (e.g.,with a fucosidase enzyme); (iii) post-translational addition of thedesired carbohydrate, e.g., after recombinant expression of anon-glycosylated glycoprotein; or (iv) purification of the glycoproteinso as to select for antibodies or antigen-binding fragments thereofwhich are not fucsoylated. See, e.g., Longmore G D & Schachter H (1982)Carbohydr Res 100: 365-92 and Imai-Nishiya H et al., (2007) BMCBiotechnol. 7: 84 for methods for producing antibodies orantigen-binding fragments thereof with no fucose content or reducedfucose content.

In some embodiments of the aspects described herein, anti-DEspRantibodies or antigen-binding fragments thereof described herein have anincreased affinity for CD32B (also known as FcγRIIB or FCGR2B), e.g., ascompared to an antibody with a wild-type Fc region, e.g., an IgG1 Fc. Insome embodiments of the aspects described herein, anti-DEspR antibodiesor antigen-binding fragments thereof described herein have a selectivelyincreased affinity for CD32B (FcγRIIB) over both CD32A (FcγRIIA) andCD16 (FcγRIIIA). Sequence alterations that result in increased affinityfor CD32B are provided, for example, in Mimoto et al., ProteinEngineering, Design & Selection 10: 589-598 (2013), Chu et al.,Molecular Immunology 45: 3926-3933 (2008), and Strohl, Current Opinionin Biology 20: 685-691 (2009), each of which is herein incorporated byreference in its entirety. In some embodiments of the aspects describedherein, the antibody or antigen-binding fragment with an increasedaffinity for CD32B comprises a heavy chain constant region, e.g., anIgG1 constant region, or fragment thereof comprising a mutation selectedfrom the group consisting of: G236D, P238D, S239D, S267E, L328F, L328E,an arginine inserted after position 236, and combinations thereof,numbered according to EU index (Kabat et al., Sequences of Proteins ofImmunological Interest, U.S. Department of Health and Human Services,Bethesda (1991)). In some embodiments of the aspects described herein,the antibody or antigen-binding fragment with an increased affinity forCD32B comprises a heavy chain constant region, e.g., an IgG1 constantregion, or fragment thereof comprising S267E and L328F substitutions. Insome embodiments of the aspects described herein, the antibody orantigen-binding fragment with an increased affinity for CD32B comprisesa heavy chain constant region, e.g., an IgG1 constant region, orfragment thereof comprising P238D and L328E substitutions. In someembodiments of the aspects described herein, the antibody orantigen-binding fragment with an increased affinity for CD32B comprisesa heavy chain constant region, e.g., an IgG1 constant region, orfragment thereof comprising a P238D substitution and substitutionselected from the group consisting of E233D, G237D, H268D, P271G, A330R,and combinations thereof. In some embodiments of the aspects describedherein, the antibody or antigen-binding fragment with an increasedaffinity for CD32B comprises a heavy chain constant region, e.g., anIgG1 constant region, or fragment thereof comprising P238D, E233D,G237D, H268D, P271G, and A330R substitutions. In some embodiments of theaspects described herein, the antibody or antigen-binding fragment withan increased affinity for CD32B comprises a heavy chain constant region,e.g., an IgG1 constant region, or fragment thereof comprising G236D andS267E. In some embodiments of the aspects described herein, the antibodyor antigen-binding fragment with an increased affinity for CD32Bcomprises a heavy chain constant region, e.g., an IgG1 constant region,or fragment thereof comprising S239D and S267E. In some embodiments ofthe aspects described herein, the antibody or antigen-binding fragmentwith an increased affinity for CD32B comprises a heavy chain constantregion, e.g., an IgG1 constant region, or fragment thereof comprisingS267E and L328F. In some embodiments of the aspects described herein,the antibody or antigen-binding fragment with an increased affinity forCD32B comprises a heavy chain constant region, e.g., an IgG1 constantregion, or fragment thereof comprising an arginine inserted afterposition 236 and L328R.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species, but inwhich the sequences of one or more of the CDR regions of V_(H) and/orV_(L) are replaced with CDR sequences of another species, such asantibodies having human heavy and light chain variable regions in whichone or more of the human CDRS (e.g., CDR3) has been replaced with mouseCDR sequences. CDR-grafted antibodies described herein comprise heavyand light chain variable region sequences from a human antibody whereinone or more of the CDR regions of V_(H) and/or V_(L) are replaced withCDR sequences of the non-human antibodies described herein, such as SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 28,SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 51, SEQ IDNO: 52, and SEQ ID NO: 53

In some embodiments of the aspects described herein, a humanizedanti-DEspR monoclonal antibody comprises mutated human IgG1, IgG2, IgG3,or IgG4 framework regions and one or more heavy and/or one or more lightchain CDR regions from the anti-human DEspR 6G8G7 or 7C5B2 variantantibodies described herein, that blocks binding of human DEspR to itsligands. In some such embodiments, the one or more variable heavy chainCDR regions comprises a sequence selected from the group consisting ofSEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23. In somesuch embodiments, the one or more variable light chain CDR regionscomprises a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 36, SEQ IDNO: 37, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 51, SEQID NO: 52, and SEQ ID NO: 53. In some such embodiments, the one or morevariable heavy chain CDR regions comprises a sequence selected from thegroup consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQID NO: 23, and the one or more variable light chain CDR regionscomprises a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 36, SEQ IDNO: 37, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 51, SEQID NO: 52, and SEQ ID NO: 53.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the aminoacid sequences of the variable heavy and light chain domains of a rodentantibody, such as that of the 6G8G7 or 7C5B2 variant antibodies (SEQ IDNO: 6, SEQ ID NO: 13, and SEQ ID NO: 20, and SEQ ID NO: 27, SEQ ID NO:34, SEQ ID NO: 41, and SEQ ID NO: 50 respectively), are screened againstthe entire library of known human variable-domain sequences, especiallygermline human variable-domain sequences. The human sequence which isclosest to that of the rodent is then accepted as the human framework(FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296(1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another methoduses a particular framework derived from the consensus sequence of allhuman antibodies of a particular subgroup of light or heavy chains. Thesame framework can be used for several different humanized antibodies(Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta etal., J. Immunol., 151:2623 (1993)).

In some embodiments of the aspects described herein, the anti-DEspRantibodies or antigen-binding fragments thereof described herein areaffinity matured or affinity optimized. Preferably, affinity optimizedanti-DEspR antibodies or antigen-binding fragments thereof haveaffinities that are at least 1-fold, at least 1.5-fold, at least 2-foldhigher, at least 5-fold higher, at least 10-fold higher, at least20-fold higher, at least 50-fold higher, or more than the 6G8G7or 7C5B2anti-DEspR variant antibodies from which they are derived.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties,for example, the anti-angiogenic properties of the 6G8G7or 7C5B2anti-DEspR variant antibodies described herein. To achieve this goal,according to a preferred method, humanized antibodies are prepared by aprocess of analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding.

Accordingly, in some embodiments, provided herein is a humanized V_(H)or variable domain of the heavy chain of a 6G8G7 HV2 variant antibody,termed herein as a “6G8G7 HV2-h1 humanized V_(H) domain” having anucleotide sequence of:

GAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGCGCCTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCCGCTACTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCGAGATCAACCCCGACTCCTCCACCATCAACTACACCCCCTCCCTGAAGGACCGCTTCACCATCTCCCGCGACACCGCCAAGAAGTCCCTGTACCTGCAGATGTCCAAGGTGCGCTCCGAGGACACCGCCCTGTACTACTGCGCCCGCCACGGCCGCGGCATGGACTACTGGTCCCAGGGCACCTCCGTGACCGTGTCCTCC (SEQ ID NO: 54); and anamino acid sequence of:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKDRFTISRDTAKKSLYLQMSKVRSEDTALYYCARHGRGMDYWSQGTSVTVSS (SEQ ID NO: 55)

In some embodiments, provided herein is a humanized V_(L) or variabledomain of the light chain of a 6G8G7 KV1 variant antibody, termed hereinas a “6G8G7 KV1-h2 humanized V_(L) domain” having a nucleotide sequenceof:

GACATCGTGCTGACCCAGTCCCCCGACATCCTGTCCGTGTCCCTGGGCGAGCGCGCCACCGTGAACTGCAAGGCCTCCCAGAACGTGGACTCCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCACCCCCCCAAGCTGCTGATCTACTCCGCCTCCTACCGCTACTCCCGCGTGCCCGACCGCATCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCAACCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTACCACTCCTACCCCCTGCTGGCCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGCGCGCCGACGCCGCCCCC (SEQ ID NO: 56); and an aminoacid sequence of:

DIVLTQSPDILSVSLGERATVNCKASQNVDSNVAWYQQKPGHPPKLLIYSASYRYSRVPDRISGSGSGTDFTLTISNLQAEDVAVYYCQQYHSYPLLAFGAGTKLELKRADAAP (SEQ ID NO: 57).

In some embodiments, provided herein is a humanized V_(L) or variabledomain of the light chain of a 6G8G7 KV2 variant antibody, termed hereinas a “6G8G7 KV2-h3 humanized V_(L) domain” having a nucleotide sequenceof:

GACATCGTGCTGACCCAGTCCCCCGACATCCTGTCCGTGTCCCTGGGCGAGCGCGCCACCGTGAACTGCAAGGCCTCCCAGAACGTGGACTCCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCACCCCCCCAAGCTGCTGATCTACTCCGCCTCCTACCGCTACTCCCGCGTGCCCGACCGCATCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCAACCTGCAGGCCGAGGACCTGGCCGACTACTTCTGCCAGCAGTACCACTCCTACCCCCTGCTGGCCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGCGCGCCGACGCCGCCCCC (SEQ ID NO: 58); and an aminoacid sequence of:

(SEQ ID NO: 59) DIVLTQSPDILSVSLGERATVNCKASQNVDSNVAWYQQKPGHPPKWYSASYRYSRVPDRISGSGSGTDFTLTISNLQAEDLADYFCQQYHSYPLLAFGAG TKLELKRADAAP

In some embodiments, provided herein is a 6G8G7 HV2-h1 human IgG1nucleotide sequence:

ATGGACCCCAAGGGCAGCCTGAGCTGGAGAATCCTGCTGTTCCTGAGCCTGGCCTTCGAGCTGAGCTACGGCGAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGCGCCTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCCGCTACTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCGAGATCAACCCCGACTCCTCCACCATCAACTACACCCCCTCCCTGAAGGACCGCTTCACCATCTCCCGCGACACCGCCAAGAAGTCCCTGTACCTGCAGATGTCCAAGGTGCGCTCCGAGGACACCGCCCTGTACTACTGCGCCCGCCACGGCCGCGGCATGGACTACTGGTCCCAGGGCACCTCCGTGACCGTGTCCTCCGCTAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGATAGTAA (SEQ ID NO:60), having an amino acid sequence of:

(SEQ ID NO: 61) MDPKGSLSWRILLFLSLAFELSYGEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKDRFTISRDTAKKSLYLQMSKVRSEDTALYYCARHGRGMDYWSQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG

In some embodiments, provided herein is a 6G8G7 HV2-h1 hinge-stabilized(Kabat S228P) human IgG4 nucleotide sequence:

ATGGACCCCAAGGGCAGCCTGAGCTGGAGAATCCTGCTGTTCCTGAGCCTGGCCTTCGAGCTGAGCTACGGCGAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGCGCCTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCCGCTACTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCGAGATCAACCCCGACTCCTCCACCATCAACTACACCCCCTCCCTGAAGGACCGCTTCACCATCTCCCGCGACACCGCCAAGAAGTCCCTGTACCTGCAGATGTCCAAGGTGCGCTCCGAGGACACCGCCCTGTACTACTGCGCCCGCCACGGCCGCGGCATGGACTACTGGTCCCAGGGCACCTCCGTGACCGTGTCCTCCGCTAGCACCAAGGGCCCCAGCGTGTTTCCTCTCGCTCCCTGCAGCCGGAGCACATCCGAGAGCACCGCTGCTCTGGGCTGTCTCGTGAAGGACTACTTCCCTGAACCCGTCACCGTCAGCTGGAATAGCGGCGCCCTGACATCCGGCGTCCACACATTCCCCGCTGTCCTGCAGAGCAGCGGCCTGTACAGCCTGAGCTCCGTGGTCACCGTGCCTAGCAGCAGCCTGGGAACAAAGACCTACACCTGCAACGTGGACCATAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAATCCAAGTATGGACCCCCCTGTCCTCCTTGCCCTGCTCCTGAATTTCTCGGAGGCCCCTCCGTCTTCCTGTTTCCCCCCAAGCCCAAGGACACCCTGATGATCTCCCGGACACCCGAAGTCACCTGCGTCGTGGTGGATGTCAGCCAGGAAGATCCCGAGGTGCAGTTCAACTGGTACGTGGACGGAGTGGAGGTGCATAACGCCAAAACCAAGCCCAGGGAAGAGCAGTTCAACAGCACCTATCGGGTCGTGTCCGTGCTCACCGTCCTGCATCAGGATTGGCTCAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCTCCTCCATCGAGAAGACCATCTCCAAGGCTAAGGGCCAACCTCGGGAGCCCCAAGTGTATACCCTCCCTCCCAGCCAGGAGGAGATGACCAAGAATCAAGTGAGCCTGACCTGCCTCGTGAAGGGATTTTACCCCTCCGACATCGCTGTGGAATGGGAAAGCAATGGCCAACCTGAGAACAACTACAAGACCACACCCCCCGTGCTGGACTCCGATGGCTCCTTCTTCCTGTACAGCAGGCTGACCGTGGACAAATCCCGGTGGCAAGAGGGAAACGTGTTCAGCTGCTCCGTGATGCACGAGGCTCTCCACAACCACTACACCCAGAAGAGCCTCTCCCTGAGCCTCGGCTAGTAA (SEQ ID NO: 62), havingan amino acid sequence of:

(SEQ ID NO: 63) MDPKGSLSWRILLFLSLAFELSYGEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKDRFTISRDTAKKSLYLQMSKVRSEDTALYYCARHGRGMDYWSQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG

In some embodiments, provided herein is a 6G8G7 HV2-h1 two amino acidvariant of SEQ ID NO: 63 having a nucleotide sequence of:

GACATCGTGCTGACCCAGTCCCCCGACATCCTGTCCGTGTCCCTGGGCGAGCGCGCCACCGTGAACTGCAAGGCCTCCCAGAACGTGGACTCCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCACCCCCCCAAGCTGCTGATCTACTCCGCCTCCTACCGCTACTCCCGCGTGCCCGACCGCATCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCAACCTGCAGGCCGAGGACCTGGCCGACTACTTCTGCCAGCAGTACCACTCCTACCCCCTGCTGGCCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGCGCGCCGACGCCGCCCCCACCGTGTCCCTGGAG (SEQ ID NO: 66),having an amino acid sequence of:

(SEQ ID NO: 67) DIVLTQSPDILSVSLGERATVNCKASQNVDSNVAWYQQKPGHPPKWYSASYRYSRVPDRISGSGSGTDFTLTISNLQAEDLADYFCQQYHSYPLLAFGAG TKLELKRADAAPTVSLE

Additional details concerning the cloning of variable heavy chaindomains with either a human IgG1 constant region or a hinge-stabilized(Kabat S228P) human IgG4 can be found in Angal, S., D. J. King, M. W.Bodmer, A. Turner, A. D. G. Lawson, G. Roberts, B. Pedley, and J. R.Adair. 1993. A single amino acid substitution abolishes theheterogeneity of chimeric mouse/human (IgG4) antibody. Mol. Immunol. 30:105-108), the contents of which are herein incorporated by reference inits entirety.

In some embodiments, provided herein is a 6G8G7 KV1-h2 human kappaconstant region nucleotide sequence:

ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACCGGAGACATCGTGCTGACCCAGTCCCCCGACATCCTGTCCGTGTCCCTGGGCGAGCGCGCCACCGTGAACTGCAAGGCCTCCCAGAACGTGGACTCCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCACCCCCCCAAGCTGCTGATCTACTCCGCCTCCTACCGCTACTCCCGCGTGCCCGACCGCATCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCAACCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTACCACTCCTACCCCCTGCTGGCCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGCGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTAA (SEQ ID NO: 64), having an amino acidsequence of:

(SEQ ID NO: 65) METDTLLLWVLLLWVPGSTGDIVLTQSPDILSVSLGERATVNCKASQNVDSNVAWYQQKPGHPPKWYSASYRYSRVPDRISGSGSGTDFTLTISNLQAEDVAVYYCQQYHSYPLLAFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

As used herein, “Vernier zone” refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which isincorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and can impact on the structure of CDRs and theaffinity of the antibody.

Known human immunoglobulin (Ig) sequences that can be used with the CDRsequences described herein are disclosed, for example, on the worldwideweb at ncbi.nlm.nih.gov/entrez-/query.fcgi; atcc.org/phage/hdb.htm1;sciquest.com/; abcam.com/; antibodyresource.com/onlinecomp.html;public.iastate.eduLabout.pedro/research_tools.htm1;mgen.uniheidelberg.de/SD/IT/IT.htm1;whfreeman.com/immunology/CH-05/kuby05.htm;library.thinkquest.org/12429/Immune/Antibody.htm1;hhmi.org/grants/lectures/1996/vlab/;path.cam.ac.uk/.about.mrc7/m-ikei-mages.htm1; antibodyresource.com/;mcb.harvard.edu/BioLinks/Immunology.htm1. immunologylink.com/;pathbox.wust1.edu/.about.hcenter/index.-htm1;biotech.ufl.edu/.about.hc1/; pebio.com/pa/340913/340913.htm1-;nal.usda.gov/awic/pubs/antibody/;m.ehime-u.acjp/.about.yasuhito-/Elisa.htm1: biodesign.com/table.asp;icnet.uk/axp/facs/davies/lin-ks.htm1;biotech.ufl.edu/.about.fccl/protocol.htm1; isac-net.org/sites_geo.htm1;aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.htm1;baserv.uci.kun.n1/.about.jraats/links1.htm1;recab.uni-hd.de/immuno.bme.nwu.edu/;mrc-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.htm1;ibt.unam.mx/virN_mice.htm1; imgt.cnusc.fr:8104/;biochem.ucl.ac.uk/.about.martin/abs/index.htm1; anti-body.bath.ac.uk/;abgen.cvm.tamu.edu/lab/wwwabgen.htm1;unizh.ch/about.honegger/AHOsem-inar/Slide01.htm1;cryst.bbk.ac.uk/.about.ubcg07s/; nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;path.cam.ac.ukhabout.mrc7/h-umanisation/TAHHP.htm1;ibt.unam.na/vir/structure/stat_aim.htm1;biosci.missouri.edu/smithgp/index.htm1;cryst.bioc.cam.ac.uk/.abo-utimolina/Web-pages/Pept/spottech.htm1;jerini.de/fr roducts.htm; patents.ibm.com/ibm.htm1.Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference. Such importedsequences can be used to reduce immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic, as known in the art.

Framework residues in the human framework regions can be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, including, but not limited to, those described in Jones et al.,Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988), Simset al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol.196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285(1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, MolecularImmunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCTpublication WO 91/09967, PCT/: U.S. patent application Ser. No.98/16280, U.S. patent application Ser. No. 96/18978, U.S. patentapplication Ser. No. 91/09630, U.S. patent application Ser. No.91/05939, U.S. patent application Ser. No. 94/01234, GB89/01334,GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246,EP 592,106; EP 519,596, EP 239,400, U.S. Pat. Nos. 5,565,332, 5,723,323,5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323,5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101,5,585,089, 5,225,539; 4,816,567, each entirely incorporated herein byreference.

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody or nucleic acid sequence providing or encoding at least80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% ofthe amino acid sequences of one or more of the framework regions. Insome embodiments, the term “acceptor” refers to the antibody amino acidor nucleic acid sequence providing or encoding the constant region(s).In yet another embodiment, the term “acceptor” refers to the antibodyamino acid or nucleic acid sequence providing or encoding one or more ofthe framework regions and the constant region(s). In a specificembodiment, the term “acceptor” refers to a human antibody amino acid ornucleic acid sequence that provides or encodes at least 80%, preferably,at least 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues not occurring at one or more specific positions of a humanantibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell-known in the art, antibodies in development, or antibodiescommercially available).

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia etal., J. Mol. Biol, 227:799 (1992), both are incorporated herein byreference). According to Chothia et al., critical portions of the CDRsof many antibodies have nearly identical peptide backbone confirmationsdespite great diversity at the level of amino acid sequence. Eachcanonical structure specifies primarily a set of peptide backbonetorsion angles for a contiguous segment of amino acid residues forming aloop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In some embodiments of thecompositions and methods described herein, the donor antibody is anantibody from a species different from the antibody from which theframework regions are obtained or derived. In the context of a humanizedantibody, the term “donor antibody” refers to a non-human antibodyproviding one or more CDRs.

As used herein, the term “key” residues refers to certain residueswithin the variable region that have more impact on the bindingspecificity and/or affinity of an antibody, in particular a humanizedantibody. A key residue includes, but is not limited to, one or more ofthe following: a residue that is adjacent to a CDR, a potentialglycosylation site (which can be either N- or O-glycosylation site), arare residue, a residue capable of interacting with the antigen, aresidue capable of interacting with a CDR, a canonical residue, acontact residue between heavy chain variable region and light chainvariable region, a residue within the Vernier zone, and a residue in theregion that overlaps between the Chothia definition of a variable heavychain CDR1 and the Kabat definition of the first heavy chain framework.

Alternatively, it is possible to produce transgenic animals (e.g., mice)that are capable, upon immunization, of producing a full repertoire ofhuman antibodies in the absence of endogenous immunoglobulin production.For example, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993);and Duchosal et al. Nature 355:258 (1992).

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S, andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

Human antibodies can also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

In some embodiments of the aspects described herein, composite humanantibody technology that generates de-immunized 100% engineered humanantibodies at the outset can be used to prepare humanized compositeanti-DEspR antibodies for use in the compositions and methods describedherein, using, for example, a technology as described by Jones, T D,Crompton U, Carr F J, Baker M P. Methods Mol Biol. 2009; 525:405-423.

Briefly, as used herein, “composite human antibodies” comprise multiplesequence segments (“composites”) derived from V-regions of unrelatedhuman antibodies that are selected to maintain monoclonal antibodysequences critical for antigen binding of the starting murine precursoranti-human DEspR monoclonal antibody, such as the 6G8G7 or 7C5B2 variantantibodies described herein, and which have all been filtered for thepresence of potential T-cell epitopes using “in silico tools” (Holgate &Baker, 2009). The close fit of human sequence segments with all sectionsof the starting antibody V regions and the elimination of CD4+T cellepitopes from the outset allow this technology to circumventimmunogenicity in the development of ‘100% engineered human’ therapeuticantibodies while maintaining optimal affinity and specificity throughthe prior analysis of sequences necessary for antigen-specificity(Holgate & Baker 2009).

As described herein, structural models of mouse anti-hDEspR antibody Vregions are produced using Swiss PDB and analyzed in order to identifyimportant “constraining” amino acids in the V regions that are likely tobe essential for the binding properties of the antibody. Residuescontained within the CDRs (using Kabat definition) together with anumber of framework residues are considered to be important. Both theV_(H) and V_(L) (V_(K)) sequences of anti-hDEspR, as described herein asSEQ ID NO: 6, SEQ ID NO: 13, and SEQ ID NO: 20, and SEQ ID NO: 27, SEQID NO: 34, SEQ ID NO: 41, and SEQ ID NO: 50, respectively, comprisetypical framework residues and the CDR1, CDR2, and CDR3 motifs arecomparable to many murine antibodies, as described elsewhere herein.

From the above analysis, it is determined that composite human sequencesof anti-hDEspR can be created with a wide latitude of alternativesoutside of CDRs but with only a narrow menu of possible alternativeresidues within the CDR sequences. Corresponding sequence segments fromseveral human antibodies can be combined to create CDRs similar oridentical to those in the murine sequences. For regions outside of andflanking the CDRs, a wide selection of human sequence segments areidentified as possible components of novel anti-DEspR composite humanantibody V regions for use with the compositions and methods describedherein.

Based upon these analyses, a large preliminary set of sequence segmentsthat can be used to create novel anti-DEspR composite human antibodyvariants are selected and analysed using ITOPE™ technology for in silicoanalysis of peptide binding to human MHC class II alleles (Perry et al2008), and using the TCED™ (T Cell Epitope Database) of known antibodysequence-related T cell epitopes (Bryson et al 2010). Sequence segmentsthat are identified as significant non-human germline binders to humanMHC class II or that scored significant hits against the TCED™ arediscarded. This results in a reduced set of segments, and combinationsof these are again analyzed, as above, to ensure that the junctionsbetween segments do not contain potential T cell epitopes. Selectedsegments are then combined to produce heavy and light chain V regionsequences for synthesis.

Accordingly, provided herein, in some aspects, are variable heavy andlight chain sequences for use in anti-DEspR composite human antibody orengineered human antibody production.

In some embodiments of the aspects described herein, an antibodyspecific for DEspR, such as, for example the anti-DEspR 6G8G7 or7C5B2variant antibodies; an anti-DEspR antibody comprising one or moreheavy chain CDR regions comprising a sequence selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ IDNO: 23; an anti-DEspR antibody comprising one or more light chain CDRregions comprises a sequence selected from the group consisting of SEQID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43,SEQ ID NO: 44, SEQ ID NO:51, SEQ ID NO: 52, and SEQ ID NO: 53; an anti-DEspR composite humanantibody comprising a variable heavy (V_(H)) chain amino acid sequenceof SEQ ID NO: 6, SEQ ID NO: 13, or SEQ ID NO: 20; an anti-DEspRcomposite human antibody comprising a variable light (V_(L)) chain aminoacid sequence of SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 41, or SEQ IDNO: 50; an anti-DEspR humanized antibody comprising a variable heavy(V_(H)) chain amino acid sequence of SEQ ID NO: 55; an anti-DEspRhumanized antibody comprising a variable light (V_(L)) chain amino acidsequence of SEQ ID NO: 57; an anti-DEspR humanized antibody comprising avariable light (V_(L)) chain amino acid sequence of SEQ ID NO: 59; ananti-DEspR humanized antibody comprising a variable heavy (V_(H)) chainIgG1 amino acid sequence of SEQ ID NO: 61; an anti-DEspR humanizedantibody comprising a variable heavy (V_(H)) chain IgG4 amino acidsequence of SEQ ID NO: 63; or an anti-DEspR humanized antibodycomprising a variable light (V_(L)) chain kappa amino acid sequence ofSEQ ID NO: 65; can be treated or processed into an antibody fragmentthereof.

Various techniques have been developed and are available for theproduction of antibody fragments. Traditionally, these fragments werederived via proteolytic digestion of intact antibodies (see, e.g.,Morimoto et al., Journal of Biochemical and Biophysical Methods24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However,these fragments can now be produced directly by recombinant host cells.For example, antibody fragments can be isolated from the antibody phagelibraries discussed above. Alternatively, Fab′-SH fragments can bedirectly recovered from E. coli and chemically coupled to form F(ab′)₂fragments (Carter et al., Bio/Technology 10:163-167 (1992)). Accordingto another approach, F(ab′)₂ fragments can be isolated directly fromrecombinant host cell culture. Other techniques for the production ofantibody fragments will be apparent to the skilled practitioner. Inother embodiments, the antibody fragment of choice is a single chain Fvfragment (scFv). See WO 93/16185.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment is a Fab fragment comprising V_(L),C_(L), V_(H) and C_(H)1 domains. Fab fragments comprise a variable andconstant domain of the light chain and a variable domain and the firstconstant domain (C_(H)1) of the heavy chain In some such embodiments,the V_(H) domain comprises, consists or consists essentially of SEQ IDNO: 6, SEQ ID NO: 13, or SEQ ID NO: 20. In some such embodiments, theV_(H) domain comprises one or more heavy chain CDR regions comprising asequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:21, SEQ ID NO: 22, and SEQ ID NO: 23. In some such embodiments, theV_(L) domain comprises, consists or consists essentially of SEQ ID NO:27, SEQ ID NO: 34, SEQ ID NO: 41, or SEQ ID NO: 50. In some suchembodiments, the V_(L) domain comprises one or more light chain CDRregions comprising a sequence selected from the group consisting of SEQID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:51, SEQ ID NO: 52, and SEQ ID NO: 53.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment is a Fab′ fragment, which is a Fabfragment having one or more cysteine residues at the C-terminus of theC_(H)1 domain.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment is a Fd fragment comprising V_(H) andC_(H)1 domains. In some such embodiments, the V_(H) domain is comprises,consists or consists essentially of SEQ ID NO: 6, SEQ ID NO: 13, or SEQID NO: 20. In some such embodiments, the V_(H) domain comprises one ormore heavy chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQID NO: 23.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment is a Fd fragment comprising V_(H) andC_(H)1 domains and one or more cysteine residues at the C-terminus ofthe C_(H)1 domain. In some such embodiments, the V_(H) domain comprises,consists or consists essentially of SEQ ID NO: 6, SEQ ID NO: 13, or SEQID NO: 20. In some such embodiments, the V_(H) domain comprises one ormore heavy chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQID NO: 23.

Single-chain Fv or scFv antibody fragments comprise the V_(H) and V_(L)domains of antibody, such that these domains are present in a singlepolypeptide chain. Generally, a Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains, which enablesthe scFv to form the desired structure for antigen binding. For a reviewof scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994). Accordingly, in some embodiments of the aspects describedherein, a human DEspR-specific antibody fragment is a Fv fragmentcomprising the V_(L) and V_(H) domains of a single arm of an antibody.In some such embodiments, the V_(H) domain comprises, consists orconsists essentially of SEQ ID NO: 6, SEQ ID NO: 13, or SEQ ID NO: 20.In some such embodiments, the V_(H) domain comprises one or more heavychain CDR regions comprising a sequence selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ IDNO: 23. In some such embodiments, the V_(L) domain comprises, consistsor consists essentially of SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 41,or SEQ ID NO: 50. In some such embodiments, the V_(L) domain comprisesone or more light chain CDR regions comprising a sequence selected fromthe group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43,SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53.

The term diabodies refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) and V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

Accordingly, in some embodiments of the aspects described herein, ahuman DEspR-specific antibody fragment is a diabody comprising twoantigen binding sites, comprising a heavy chain variable domain (V_(H))connected to a light chain variable domain (V_(L)) in the samepolypeptide chain. In some such embodiments, the V_(H) domain comprises,consists or consists essentially of SEQ ID NO: 6, SEQ ID NO: 13, or SEQID NO: 20. In some such embodiments, the V_(H) domain comprises one ormore heavy chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQID NO: 23. In some such embodiments, the V_(L) domain comprises,consists or consists essentially of SEQ ID NO: 27, SEQ ID NO: 34, SEQ IDNO: 41, or SEQ ID NO: 50. In some such embodiments, the V_(L) domaincomprises one or more light chain CDR regions comprising a sequenceselected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQID NO: 30, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 42,SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ IDNO: 53.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment is a dAb fragment comprising a V_(H)domain. In some such embodiments, the V_(H) domain comprises, consistsor consists essentially of SEQ ID NO: 6, SEQ ID NO: 13, or SEQ ID NO:20. In some such embodiments, the V_(H) domain comprises one or moreheavy chain CDR regions comprising a sequence selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ IDNO: 23.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment comprises isolated CDR regions. In somesuch embodiments, the isolated CDR region comprises one or more heavychain CDR regions comprising a sequence selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ IDNO: 23. In some such embodiments, the isolated CDR region comprises oneor more light chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43, SEQID NO: 44, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53.

In some embodiments of the aspects described herein, the humanDEspR-specific antibody fragment is a F(ab′)₂ fragment, which comprisesa bivalent fragment comprising two Fab′ fragments linked by a disulphidebridge at the hinge region.

“Linear antibodies” refer to the antibodies as described in Zapata etal., Protein Eng., 8(10):1057-1062 (1995). Briefly, these antibodiescomprise a pair of tandem Fd segments (V_(H)—C_(H)1-V_(H)—C_(H)1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

In some embodiments of the aspescts described herein, a humanDEspR-specific antibody fragment is a linear antibody comprising a pairof tandem Fd segments (V_(H)—C_(H)1-V_(H)—C_(H)1) which, together withcomplementary light chain polypeptides, form a pair of antigen bindingregions. In some such embodiments, the V_(H) domain comprises, consistsor consists essentially of SEQ ID NO: 6, SEQ ID NO: 13, or SEQ ID NO:20. In some such embodiments, the V_(H) domain comprises one or moreheavy chain CDR regions comprising a sequence selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ IDNO: 23. In some such embodiments, the V_(L) domain consists or consistsessentially of SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 41, or SEQ IDNO: 50. In some such embodiments, the V_(L) domain comprises one or morelight chain CDR regions comprising a sequence selected from the groupconsisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43, SEQ IDNO: 44, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53.

In some embodiments of these aspects, a human DEspR-specific antibodyfragment has specificity for the same epitope as a monoclonal anti-DEspRantibody 6G8G7 variant, described herein, and produced by hybridoma6G8G7, or alternatively the specificity for the same epitope as amonoclonal anti-DEspR antibody 7C5B2 variant, described herein, andproduced by hybridoma 7C5B2. In some embodiments of these aspects, ahuman DEspR-specific antigen-binding fragment has specificity for anepitope comprising, consisting essentially of, or consisting of SEQ IDNO: 1. In some embodiments of these aspects, a human DEspR-specificantigen-binding fragment has specificity for an epitope comprising,consisting essentially of, or consisting of SEQ ID NO: 2.

Examples of DEspR-inhibiting antibodies are described inPCT/US2005/041594, PCT/US2011/045056, and PCT/US2013/022537, thecontents of each of which are incorporated herein by reference in theirentireties.

In some embodiments of the aspects described herein, a DEspR bindingprotein, an isolated antibody or antigen-binding fragment thereof, oranti-DEspR antibody or antigen-binding fragment thereof as disclosedherein binds to DEspR present on intact cells or an antigenic-pepitdecontaining a DEspR epitope, such as an epitope of SEQ ID NOs: 1 or 2,with a EC50 of 12 μg/ml or less, 10 μg/ml or less, 7 μg/ml or less, 5μg/ml or less, 3 μg/ml or less, 1.5 μg/ml or less, 1.2 μg/ml or less, 1μg/ml or less, 0.7 μg/ml or less, or 0.64 μg/ml or less (see, e.g.,Tables 2 and 5). Preferably, the antibody or antigen-binding fragmentthereof is a neutralizing antibody or a DEspR antagonist. In someembodiments of the aspects described herein, a DEspR binding protein,isolated antibody or antigen-binding fragment thereof, or anti-DEspRantibody or antigen-binding fragment thereof as disclosed herein bindsto DEspR present on intact cells or an antigenic-pepitde containing aDEspR epitope, such as an epitope of SEQ ID NOs: 1 or 2, with an EC50 of30 nM or less, an EC50 of 25 nM or less, an EC50 of 24 nM or less, anEC50 of 23 nM or less, an EC50 of 21 nM or less, an EC50 of 20 nM orless, or an EC50 of 15 nM or less (see, e.g., see Table 2 and 5).Preferably, the antibody or antigen-binding fragment thereof is aneutralizing antibody or a DEspR antagonist. The EC50 can be determined,for example, by measuring the binding of one of the described DEspRantibodies or antigen-binding fragments thereof to an antigenic peptidecontaining the epitope such as SEQ ID NO. 1 or SEQ ID NO: 2, by, forexample, ELISA (see, e.g., Table 1) or to the receptor on an intacthuman cell (e.g. tha is positive for DEspR by FACS (see e.g. Table 2,FIG. 37).

In some embodiments of the aspects described herein, a DEspR bindingprotein, an isolated antibody or antigen-binding fragment thereof, oranti-DEspR antibody or antigen-binding fragment thereof as disclosedherein has an IC50 of 3.0 μg/ml or less, an IC50 of 2.8 μg/ml or less,an IC50 of 2.6 μg/ml or less, an IC50 of 2.5 μg/ml or less, an IC50 of2.0 μg/ml or less, an IC50 of 1.5 μg/ml or less, an IC50 of 1.2 μg/ml orless, or an IC50 of 1.0 μg/ml or less, as determined by, for example,inhibition of activated neutrophil survival or human angiogenesis assaysas descrived herein (see, for example, Tables 3-5 herein). IC50 can bedetermined, for example, by measuring the ability of one of thedescribed DEspR antibodies or antigen-binding fragments thereof toinhibit neutrophil survival (see, e.g., Tables 3, 5) or in inhibitingbFGF-medicated NEGF-independent angiogenesis of human umbilical veincells (see, e.g., Table 4, FIGS. 3, 9 and 40). Neutrophil survival canbe performed with freshly isolated rat neutrophils. For example,neutrophils (e.g., 50,000/well) are incubated in the presence or absenceof the antibody at 37° C. for 4 hours and live cells counted by using,for example, Tryphan blue. Any of the standard HUVEC angiogenesis assaysknown to one of ordinary skill in the art can be used.

In some embodiments of the aspects described herein, a DEspR bindingprotein, an isolated antibody or antigen-binding fragment thereof, oranti-DEspR antibody or antigen-binding fragment thereof as disclosedherein has a K_(D) of less than 5.0 μg/ml, less than 4.0 μg/ml, lessthan 3 μg/ml, less than 2.5 μm/ml, less than 2.0 μm/ml, less than 1.5μg/ml, or less than 1.0 μg/ml, for binding to DEspR+ pancreatic cancercells. In some embodiments, a DEspR binding protein, an isolatedantibody or antigen-binding fragment thereof, or anti-DEspR antibody orantigen-binding fragment thereof as disclosed herein has a K_(D) of lessthan 35 nM, less than 33 nM, less than 30 nM, or between 15-35 nM forbinding to DEspR+ pancreatic cancer cells. In some embodiments of theaspects described herein, a DEspR binding protein, an isolated antibodyor antigen-binding fragment thereof, or anti-DEspR antibody orantigen-binding fragment thereof as disclosed herein has a of less than5.0 μg/ml, less than 4.0 μg/ml, less than 3 μg/ml, less than 2.5 μm/ml,less than 2.0 μm/ml, less than 1.5 μg/ml, or less than 1.0 μg/ml, forbinding to DEspR+ pancreatic cancer cells, or binding to SEQ ID NO: 2and is a neutralizing antibody or DEspR antagonist.

In some embodiments of the aspects described herein, a DEspR bindingprotein, an isolated antibody or antigen-binding fragment thereof, oranti-DEspR antibody or antigen-binding fragment thereof as disclosedherein has at least of a EC50 of 12 μg/ml less, an IC50 of 3.0 μg/ml, orless, or a K_(D) of 5.2 μg/ml or less. In some embodiments, a DEspRbinding protein, an isolated antibody or antigen-binding fragmentthereof, or anti-DEspR antibody or antigen-binding fragment thereof asdisclosed herein has a EC50 of 12 μg/ml or less, and an IC50 of 3.0μg/ml or less, and a K_(D) of 5.2 μg/ml or less and is a neutralizingantibody or DEspR antagonist.

In some embodiments of the aspects described herein, amino acid sequencemodification(s) of the antibodies or antigen-binding fragments thereofspecific for DEspR, such as the 6G8G7 or 7C5B2 variant antibodies or anantigen-binding fragments thereof described herein are contemplated. Forexample, it can be desirable to improve the binding affinity and/orother biological properties of the antibody. Amino acid sequencevariants of the antibody are prepared by introducing appropriatenucleotide changes into the antibody nucleic acid, or by peptidesynthesis. Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of the antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics,e.g., binding specificity, inhibition of biological activity. The aminoacid changes also can alter post-translational processes of theantibody, such as changing the number or position of glycosylationsites.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includeantibody with an N-terminal methionyl residue or the antibody fused to acytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated for use in the antibodies orantigen-binding fragments thereof specific for DEspR described herein.

Substantial modifications in the biological properties of the antibodiesor antigen-binding fragments thereof specific for DEspR are accomplishedby selecting substitutions that differ significantly in their effect onmaintaining (a) the structure of the polypeptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain. Amino acids can be grouped according tosimilarities in the properties of their side chains (in A. L. Lehninger,in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York(1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe(F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T),Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4)basic: Lys (K), Arg (R), His (H).

Alternatively, naturally occurring residues can be divided into groupsbased on common side-chain properties: (1) hydrophobic: Norleucine, Met,Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;(3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues thatinfluence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibodies or antigen-binding fragments thereof specific forDEspR also can be substituted, generally with serine, to improve theoxidative stability of the molecule and prevent aberrant crosslinkingConversely, cysteine bond(s) can be added to the antibody to improve itsstability (particularly where the antibody is an antigen-bindingfragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g., the monoclonal 6G8G7 or 7C5B2 anti-DEspR variantantibodies, or a humanized or human antibody or antigen-binding fragmentthereof specific for DEspR, as provided herein). Generally, theresulting variant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding.

Alternatively, or additionally, it can be beneficial to analyze acrystal structure of the antigen-antibody complex to identify contactpoints between the antibody or antigen-binding fragments thereofspecific for DEspR and human DEspR. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies or antigen-binding fragments thereof with superior propertiesin one or more relevant assays can be selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine can also be used.

Addition of glycosylation sites to the antibodies or antigen-bindingfragments thereof specific for DEspR is accomplished by altering theamino acid sequence such that it contains one or more of theabove-described tripeptide sequences (for N-linked glycosylation sites).The alteration can also be made by the addition of, or substitution by,one or more serine or threonine residues to the sequence of the originalantibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto can be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in U.S. Pat Appl No. US 2003/0157108 Al, Presta,L. See also US 2004/0093621 Al (Kyowa Hakko Kogyo Co., Ltd). Antibodieswith a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrateattached to an Fc region of the antibody are referenced in WO03/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO97/30087, Patel et al.See, also, WO98/58964 (Raju, S.) and WO99/22764 (Raju, S.) concerningantibodies with altered carbohydrate attached to the Fc region thereof.

In some embodiments, it can be desirable to modify the antibodies orantigen-binding fragments thereof specific for DEspR described hereinwith respect to effector function, e.g., so as to enhanceantigen-dependent cell-mediated cyotoxicity (ADCC) and/or complementdependent cytotoxicity (CDC) of the antibody. This can be achieved byintroducing one or more amino acid substitutions in an Fc region of theantibody or antigen-binding fragment thereof. Alternatively oradditionally, cysteine residue(s) can be introduced in the Fc region,thereby allowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated can have improved internalizationcapability and/or increased complement-mediated cell killing andantibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J.Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922(1992). Homodimeric antibodies with enhanced anti-tumor activity canalso be prepared using heterobifunctional cross-linkers as described inWolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, anantibody can be engineered which has dual Fc regions and can therebyhave enhanced complement lysis and ADCC capabilities. See Stevenson etal. Anti-Cancer Drug Design 3:219-230 (1989).

For example, WO00/42072 (Presta, L.) describes antibodies with improvedADCC function in the presence of human effector cells, where theantibodies comprise amino acid substitutions in the Fc region thereof.Preferably, the antibody with improved ADCC comprises substitutions atpositions 298, 333, and/or 334 of the Fc region (Eu numbering ofresidues). Preferably the altered Fc region is a human IgG1 Fc regioncomprising or consisting of substitutions at one, two or three of thesepositions. Such substitutions are optionally combined withsubstitution(s) which increase C1q binding and/or CDC.

Antibodies with altered Clq binding and/or complement dependentcytotoxicity (CDC) are described in WO99/51642, U.S. Pat. No.6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No. 6,528,624B1 andU.S. Pat. No. 6,538,124 (Idusogie et al.). The antibodies comprise anamino acid substitution at one or more of amino acid positions 270, 322,326, 327, 329, 313, 333 and/or 334 of the Fc region thereof (Eunumbering of residues).

To increase the serum half life of the antibody specific for DEspRdescribed herein, one can incorporate a salvage receptor binding epitopeinto the antibody (especially an antigen-binding fragment) as describedin U.S. Pat. No. 5,739,277, for example. As used herein, the term“salvage receptor binding epitope” refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule.

Antibodies with improved binding to the neonatal Fc receptor (FcRn), andincreased half-lives, are described in WO00/42072 (Presta, L.) andUS2005/0014934A1 (Hinton et al.). These antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. For example, the Fc region can have substitutions at oneor more of positions 238, 250, 256, 265, 272, 286, 303, 305, 307, 311,312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428 or434 (EU numbering of residues). The preferred Fc region-comprisingantibody variant with improved FcRn binding comprises amino acidsubstitutions at one, two or three of positions 307, 380 and 434 of theFc region thereof (EU numbering of residues). In one embodiment, theantibody has 307/434 mutations.

Engineered antibodies specific for DEspR with three or more (preferablyfour) functional antigen binding sites are also contemplated (US ApplnNo. US2002/0004587 Al, Miller et al.).

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

In some embodiments of the aspects described herein, antibody andantigen-binding fragments specific for DEspR described herein areincorporated together with one or more other antibody andantigen-binding fragments specific for other targets to produce “dualtargeting” or multi-targeting molecules which bind to one or moretargets additional to DEspR. Examples of dual targeting strategies arereviewed in Kontermann (MAbs. 2012 Mar-Apr; 4(2): 182-197).

DEspR Binding Proteins

In various embodiments, provided herein are DEspR Binding Proteins,e.g., DVD-Ig binding proteins that bind one or more epitopes of DEspRpolypeptide. An exemplary embodiment of such DVD-Ig molecules comprisesa heavy chain that comprises the structural formulaVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain variabledomain, VD2 is a second heavy chain variable domain, C is a heavy chainconstant domain, X1 is a linker, X2 is an Fc region on the firstpolypeptide chain and X2 does not comprise an Fc region on the secondpolypeptide chain; n is independently 0 or 1 on the first and secondchains; and a light chain that comprises the structural formulaVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variabledomain, VD2 is a second light chain variable domain, C is a light chainconstant domain, X1 is a linker, and X2 is an Fc region on the firstpolypeptide chain and X2 does not comprise an Fc region on the secondpolypeptide chain; n is independently 0 or 1 on the first and secondchains. Such a DVD-Ig may comprise two such heavy chains and two suchlight chains, wherein each chain comprises variable domains linked intandem without an intervening constant region between the variabledomains, wherein a heavy chain and a light chain associate to form twotandem antigen binding sites, and a pair of heavy and light chains mayassociate with another pair of heavy and light chains to form atetrameric binding protein with four antigen binding sites. In anotherembodiment, a DVD-Ig molecule may comprise heavy and light chains thateach comprise three variable domains, e.g., VD1, VD2, VD3, linked intandem without an intervening constant region between variable domains,wherein a pair of heavy and light chains may associate to form threeantigen binding sites, and wherein a pair of heavy and light chains mayassociate with another pair of heavy and light chains to form atetrameric binding protein with six antigen binding sites.

Each variable domain (VD) in a DVD-Ig may be obtained from one or more“parent” monoclonal antibodies that bind one or more desired antigens orepitopes, such as LRP-8 antigens or epitopes. General methods of makingDVD-Ig and properties associated with DVD-Igs are described in U.S. Pat.No. 8,841,417, incorporated by reference herein in its entirety.

Antibody and Binding-Protein Conjugates:

In some embodiments of the aspects described herein, immunoconjugatescomprising a DEspR-binding protein, or anti-DEspR antibody orantigen-binding fragments specific for DEspR described herein areconjugated to an agent such as a chemotherapeutic agent, toxin (e.g. anenzymatically active toxin of bacterial, fungal, plant or animal origin,or fragments thereof), a small molecule, an siRNA, a nanoparticle, atargeting agent (e.g., a microbubble), or a radioactive isotope (i.e., aradioconjugate) can be used. Such immunoconjugates can be used, forexample, in diagnostic, theranostic, or targeting methods.

In some embodiments, antibody or binding-protein drug conjugatescomprising DEspR-binding protein, or anti-DEspR antibody orantigen-binding fragments specific for DEspR described herein areprovided. The terms “antibody drug conjugate” or “antibody-drugconjugate,” as used herein, refer to an antibody conjugated to anon-proteinaceous agent, typically a chemotherapeutic agent, e.g., acytotoxic agent, a cytostatic agent, a toxin, or a radioactive agent. Alinker molecule can be used to conjugate the drug to the antibody. Awide variety of linkers and drugs useful in ADC technology are known inthe art and can be used in embodiments described herein. (See, forexample, US20090028856; US2009/0274713; US2007/0031402; WO2005/084390;WO2009/099728; U.S. Pat. No. 5,208,020; U.S. Pat. No. 5,416,064; U.S.Pat. Nos. 5,475,092; 5,585,499; 6,436,931; 6,372,738; and 6,340,701, allincorporated herein by reference in their entireties). By combining theunique targeting of monoclonal antibodies or fragments thereof with thecancer-killing ability of cytotoxic drugs, antibody drug conjugatesallow sensitive and increased discrimination between healthy anddiseased tissue.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates are described herein. Enzymatically active toxins andfragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugate antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and¹⁸⁶Re.

Conjugates of the antibodies specific for DEspR described herein and acytotoxic agent can be made using any of a variety of bifunctionalprotein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Forexample, a ricin immunotoxin can be prepared as described in Vitetta etal. Science 238: 1098 (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See WO94/11026.

In some embodiments, the DEspR-specific antibody or antigen-bindingfragment thereof, such as the 6G8G7 or 7C5B2 variant antibodies orantigen-binding fragments thereof, or a humanized or composite antibodyor antgen-binding fragment thereof derived or obtained from the 6G8G7 or7C5B2 variant antibodies, can be conjugated to a “receptor” (such as,for example, streptavidin) for utilization in tumor pretargeting whereinthe antibody-receptor conjugate is administered to the subject, followedby removal of unbound conjugate from the circulation using a clearingagent and then administration of a “ligand” (e.g. avidin) which isconjugated to a cytotoxic agent (e.g. a radionucleotide). In someembodiments, the DEspR-specific antibody or antigen-binding fragmentthereof can be conjugated to biotin, and the biotin conjugated antibodyor antigen-binding fragment thereof can be further conjugated or linkedto a streptavidin-bound or -coated agent, such as a streptavidin-coatedmicrobubble, for use in, for example, molecular imaging of angiogenesis.

The antibodies and antigen-binding fragments thereof specific for DEspRdescribed herein, such as the 6G8G7 or 7C5B2 variant antibodies orantigen-binding fragments thereof, or a humanized or composite antibodyor antgen-binding fragment thereof derived or obtained from the 6G8G7 or7C5B2 variant antibodies, can also be formulated as immunoliposomes.Liposomes containing the antibody are prepared by methods known in theart, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA,82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030(1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated, for example, by thereverse phase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

The hybridoma cell lines 6G8G7 amd 7C5B2 are being maintained andstored.

Therapeutic & Diganotic Uses of DEsR Binding Proteins, Anti-DEspRAntibodies and Fragments Thereof

As described herein, the inventors have discovered that both the 7C5B2and 6G8G7 anti-DEspR variant antibodies, and fully humanized antibodyderivatives thereof inhibit cancer stem cell growth and anoikisresistance in multiple human cancer cell lines and decreases tumorprogression and increases survival using a pancreatic peritonealmetastasis nude rat model. In addition, the data provided hereindemonstrate that the 6G8G7 anti-DEspR antibody decreases tumorinitiation/tumorigenesis of Panc1-CSCs, decreases collagen-1 (col1)secretion by Panc1-CSCs, and decreases αSMA expression induced by TNF-α.

Accordingly, provided herein, in some aspects, are methods of treatingan angiogenesis-dependent disease or disorder comprising administering atherapeutically effective amount of an antibody or antigen-bindingspecific for DEspR. Such methods can comprise administering, forexample, a chimeric, humanized, deimmunized, or composite humananti-DEspR antibody derived from the 6G8G7 or 7C5B2 variant antibodies;an anti-DEspR antibody comprising one or more heavy chain CDR regionscomprising a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23; an anti-DEspRantibody comprising one or more light chain CDR regions comprising asequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ IDNO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 52, andSEQ ID NO: 53; an anti-DEspR composite human antibody comprising avariable heavy (VH) chain amino acid sequence consisting of SEQ ID NO:6, SEQ ID NO: 13, or SEQ ID NO: 20; an anti-DEspR composite humanantibody comprising a variable light (VL) chain amino acid sequenceconsisting of SEQ ID NO: 27, SEQ ID NO: 34, or SEQ ID NO: 41; ananti-DEspR antibody comprising one or more CDRs, e.g. 1 CDR, 2 CDRs, 3CDRs, 4 CDRs, 5 CDRs, or 6 CDRs, selected from the group consisting of(a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 7,SEQ ID NO: 14, or SEQ ID NO: 21; (b) a heavy chain CDR2 having the aminoacid sequence of SEQ ID NO: 8, SEQ ID NO: 15, or SEQ ID NO: 22; (c) aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 9; SEQ IDNO: 16, or SEQ ID NO: 22; (d) a light chain CDR1 having the amino acidsequence of SEQ ID NO: 28, SEQ ID NO: 35, or SEQ ID NO: 42; (e) a lightchain CDR2 having the amino acid sequence of SEQ ID NO: 29, SEQ ID NO:36, or SEQ ID NO: 43; and (f) a light chain CDR3 having the amino acidsequence of SEQ ID NO: 30, SEQ ID NO: 37, or SEQ ID NO: 44; ananti-DEspR antibody comprising a heavy chain or a fragment thereof,comprising one or more CDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs, selectedfrom the group consisting of a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21; a heavy chainCDR2 having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 15, orSEQ ID NO: 22; and a heavy chain CDR3 having the amino acid sequence ofSEQ ID NO: 9; SEQ ID NO: 16, or SEQ ID NO: 22; an anti-DEspR antibodycomprising a light chain or a fragment thereof, comprising one or moreCDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs selected from the group consistingof a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28,SEQ ID NO: 35, or SEQ ID NO: 42; a light chain CDR2 having the aminoacid sequence of SEQ ID NO: 29, SEQ ID NO: 36, or SEQ ID NO: 43; and alight chain CDR3 having the amino acid sequence of SEQ ID NO: 30, SEQ IDNO: 37, or SEQ ID NO: 44.

These antiangiogenic therapies can be used as cancer treatmentstrategies aimed at inhibiting existing tumor blood vessels anddevelopment of tumor blood vessels required for providing nutrients tosupport tumor growth. Because angiogenesis is involved in both primarytumor growth and metastasis, the antiangiogenic treatments using theantibodies and antigen-binding fragments specific for DEspR describedherein are capable of inhibiting the neoplastic growth of tumor at theprimary site, as well as preventing micro- and macro-metastasis oftumors at the secondary sites, therefore allowing attack of the tumorsby other therapeutics. Angiogenesis-dependent diseases and disordersthat can be treated using the methods and compositions described hereinare those diseases and disorders affected by vascular growth. In otherwords, an “angiogenesis-dependent disease or disorder” refers to thosediseases or disorders that are dependent on a rich blood supply andblood vessel proliferation for the diseases' pathological progression(e.g., metastatic tumors), or diseases or disorders that are the directresult of aberrant blood vessel proliferation (e.g., diabeticretinopathy and hemangiomas).

In some aspects, also provided herein, are methods of treating cancer ortumor metastasis comprising administering a therapeutically effectiveamount of an antibody or antigen-binding fragment thereof specific forDEspR. Such methods can comprise administering, for example, a chimeric,humanized, deimmunized, or composite human anti-DEspR antibody derivedfrom the 6G8G7 or variant 7C5B2 antibody; an anti-DEspR antibodycomprising one or more heavy chain CDR regions comprising a sequenceselected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQID NO: 22, and SEQ ID NO: 23; an anti-DEspR antibody comprising one ormore light chain CDR regions comprises a sequence selected from thegroup consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43,SEQID NO: 44, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53; ananti-DEspR composite human antibody comprising a variable heavy (V_(H))chain amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 13, or SEQ ID NO:20; an anti-DEspR composite human antibody comprising a variable light(V_(L)) chain amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 34, SEQID NO: 41, or SEQ ID NO: 50; an anti-DEspR antibody comprising one ormore CDRs, e.g. 1 CDR, 2 CDRs, 3 CDRs, 4 CDRs, 5 CDRs, or 6 CDRs,selected from the group consisting of (a) a heavy chain CDR1 having theamino acid sequence of SEQ ID NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21;(b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8,SEQ ID NO: 15, or SEQ ID NO: 22; (c) a heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 9; SEQ ID NO: 16, or SEQ ID NO: 23; (d) alight chain CDR1 having the amino acid sequence of SEQ ID NO: 28, SEQ IDNO: 35, SEQ ID NO: 42, or SEQ ID NO: 51; (e) a light chain CDR2 havingthe amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 43,or SEQ ID NO: 52; and (f) a light chain CDR3 having the amino acidsequence of SEQ ID NO: 30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ ID NO:53; an anti-DEspR antibody comprising a heavy chain or a fragmentthereof, comprising one or more CDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs,selected from the group consisting of a heavy chain CDR1 having theamino acid sequence of SEQ ID NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21; aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8, SEQ IDNO: 15, or SEQ ID NO: 22; and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 9; SEQ ID NO: 16, or SEQ ID NO: 22; an anti-DEspRantibody comprising a light chain or a fragment thereof, comprising oneor more CDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs selected from the groupconsisting of a light chain CDR1 having the amino acid sequence of SEQID NO: 28, SEQ ID NO: 35, SEQ ID NO: 42, or SEQ ID NO: 51; a light chainCDR2 having the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 36, SEQID NO: 43, or SEQ ID NO: 52; and a light chain CDR3 having the aminoacid sequence of SEQ ID NO: 30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ IDNO: 53; an anti-DEspR humanized antibody comprising a variable heavy(VH) chain amino acid sequence of SEQ ID NO: 55; an anti-DEspR humanizedantibody comprising a variable light (V_(L)) chain amino acid sequenceof SEQ ID NO: 57; an anti-DEspR humanized antibody comprising a variablelight (V_(L)) chain amino acid sequence of SEQ ID NO: 59; an anti-DEspRhumanized antibody comprising a variable heavy (V_(H)) chain IgG1 aminoacid sequence of SEQ ID NO: 61; an anti-DEspR humanized antibodycomprising a variable heavy (V_(H)) chain IgG4 amino acid sequence ofSEQ ID NO: 63; or an anti-DEspR humanized antibody comprising a variablelight (V_(L)) chain kappa amino acid sequence of SEQ ID NO: 65. Suchanti-metastasis thereapies provide cancer treatment strategies aimed atinhibiting concurrent inhibition of tumor vascularization and tumor cellinvasiveness for treatment and/or inhibition of micrometastasis andmacrometastasis, as further described herein. Furthermore, since DEspRis also expressed in tumor cells, including cancer stem cells, asdemonstrated herein, immunoconjugates of DEspR specific antibodies orantigen-binding fragments thereof, as described herein, can be generatedby conjugation to any agent such as a toxin, cytotoxic or pro-apoptoticagent, and can further inhibit tumor growth by directlytargeting/killing tumor cells and cancer stem cells.

Angiogenesis is a process of tissue vascularization that involves boththe growth of new developing blood vessels into a tissue(neo-vascularization) and co-opting of existing blood vessels to atarget site. Blood vessels are the means by which oxygen and nutrientsare supplied to living tissues and waste products are removed fromliving tissue. Angiogenesis can be a critical biological process. Forexample, angiogenesis is essential in reproduction, development andwound repair. Conversely, inappropriate angiogenesis can have severenegative consequences. For example, it is only after solid tumors arevascularized as a result of angiogenesis that the tumors have asufficient supply of oxygen and nutrients that permit it to grow rapidlyand metastasize.

Where the growth of new blood vessels is the cause of, or contributesto, the pathology associated with a disease, inhibition of angiogenesis,using the compositions and methods described herein, can reduce thedeleterious effects of the disease. Non-limiting examples includetumors, carotid artery disease, rheumatoid arthritis, diabeticretinopathy, inflammatory diseases, restenosis, and the like. Where thegrowth of new blood vessels is required to support growth of adeleterious tissue, inhibition of angiogenesis, using the compositionsand methods described herein, can reduce the blood supply to the tissueand thereby contribute to reduction in tissue mass based on blood supplyrequirements. Non-limiting examples include growth of tumors whereneovascularization is a continual requirement in order that the tumorgrowth beyond a few millimeters in thickness, and for the establishmentof solid tumor metastases. Another example is coronary plaqueenlargement.

There are a variety of diseases or disorders in which angiogenesis isbelieved to lead to negative consequences, referred to herein as“angiogenesis-dependent disease or disorder,” “pathologicalangiogenesis,” or “diseases or disorders dependent or modulated byangiogenesis,” including but not limited to, inflammatory disorders suchas immune and non-immune inflammation, chronic articular rheumatism andpsoriasis, disorders associated with inappropriate or inopportuneinvasion of vessels such as diabetic retinopathy, neovascular glaucoma,restenosis, capillary proliferation in atherosclerotic plaques andosteoporosis, and cancer associated disorders, such as solid tumors,solid tumor metastases, angiofibromas, retrolental fibroplasia,hemangiomas, Kaposi sarcoma and the like cancers which requireneovascularization to support tumor growth. In some embodiments of theaspects described herein, the methods are directed to inhibitingangiogenesis in a subject with cancer.

Non-limiting examples of angiogenesis-dependent diseases or disordersthat can be treated using the compositions and methods described hereininclude abnormal vascular proliferation, ascites formation, psoriasis,age-related macular degeneration, thyroid hyperplasia, preeclampsia,rheumatoid arthritis and osteoarthritis, carotid artery disease, vasovasorum neovascularization, vulnerable plaque neovascularization,neurodegenerative disorders, Alzheimer's disease, obesity, pleuraleffusion, atherosclerosis, endometriosis, diabetic/other retinopathies,ocular neovascularizations, such as neovascular glaucoma and cornealneovascularization, disorders associated with inappropriate orinopportune invasion of vessels such as diabetic retinopathy, maculardegeneration, neovascular glaucoma, restenosis, capillary proliferationin atherosclerotic plaques and osteoporosis, and cancer associateddisorders, such as solid tumors, solid tumor metastases, angiofibromas,retrolental fibroplasia, hemangiomas, Kaposi sarcoma, cancers whichrequire neovascularization to support tumor growth, etc.

Accordingly, described herein are methods of inhibiting angiogenesis ina tissue of a subject or individual having a disease or disorderdependent or modulated by angiogenesis, where the disease or disordercan be treated by the inhibition of angiogenesis. Generally, the methodscomprise administering to the subject a therapuetically effective amountof a composition comprising an angiogenesis-inhibiting amount of ananti-DEspR antibody or antigen-binding fragment thereof, as describedherein. In some embodiments of the methods described herein, the methodsfurther comprises selecting or diagnosing a subject having or at riskfor a disease or disorder dependent on angiogenesis.

In some embodiments of these methods, the DEspR is human DEspR. In someembodiments of these methods, the DEspR target has a sequence comprisingSEQ ID NO: 3 or an allelic variant thereof. In some embodiments of thesemethods, an antibody or antigen-binding fragment thereof thatspecifically binds to DEspR and inhibits DEspR biological activityblocks interaction of DEspR with VEGFsp. In some embodiments of thesemethods, the VEGFsp has a sequence comprising the sequence of SEQ ID NO:4. In some embodiments of these methods, the antibody or antigen-bindingfragment thereof is specific for an epitope of DEspR comprising anextracellular portion of DEspR. In some embodiments of these methods,the antibody or antigen-binding fragment thereof is specific for anepitope of DEspR comprising, consisting essentially of, or consisting ofSEQ ID NO: 1. In some embodiments of these methods, the antibody orantigen-binding fragment thereof is specific for an epitope of DEspRcomprising, consisting essentially of, or consisting of SEQ ID NO: 2.

In some embodiments of these methods for inhibiting angiogenesis ortreating an angiogenesis-dependent disease or disorder, the antibody orantigen-binding fragment thereof specific for DEspR is a a chimeric,humanized, deimmunized, or composite human anti-DEspR antibody derivedfrom the 6G8G7 or variant 7C5B2 antibody; an anti-DEspR antibodycomprising one or more heavy chain CDR regions comprising a sequenceselected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQID NO: 22, and SEQ ID NO: 23; an anti-DEspR antibody comprising one ormore light chain CDR regions comprises a sequence selected from thegroup consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43,SEQID NO: 44, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53; ananti-DEspR composite human antibody comprising a variable heavy (V_(H))chain amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 13, or SEQ ID NO:20; an anti-DEspR composite human antibody comprising a variable light(V_(L)) chain amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 34, SEQID NO: 41, or SEQ ID NO: 50; an anti-DEspR antibody comprising one ormore CDRs, e.g. 1 CDR, 2 CDRs, 3 CDRs, 4 CDRs, 5 CDRs, or 6 CDRs,selected from the group consisting of (a) a heavy chain CDR1 having theamino acid sequence of SEQ ID NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21;(b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8,SEQ ID NO: 15, or SEQ ID NO: 22; (c) a heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 9; SEQ ID NO: 16, or SEQ ID NO: 23; (d) alight chain CDR1 having the amino acid sequence of SEQ ID NO: 28, SEQ IDNO: 35, SEQ ID NO: 42, or SEQ ID NO: 51; (e) a light chain CDR2 havingthe amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 43,or SEQ ID NO: 52; and (f) a light chain CDR3 having the amino acidsequence of SEQ ID NO: 30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ ID NO:53; an anti-DEspR antibody comprising a heavy chain or a fragmentthereof, comprising one or more CDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs,selected from the group consisting of a heavy chain CDR1 having theamino acid sequence of SEQ ID NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21; aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8, SEQ IDNO: 15, or SEQ ID NO: 22; and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 9; SEQ ID NO: 16, or SEQ ID NO: 22; an anti-DEspRantibody comprising a light chain or a fragment thereof, comprising oneor more CDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs selected from the groupconsisting of a light chain CDR1 having the amino acid sequence of SEQID NO: 28, SEQ ID NO: 35, SEQ ID NO: 42, or SEQ ID NO: 51; a light chainCDR2 having the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 36, SEQID NO: 43, or SEQ ID NO: 52; and a light chain CDR3 having the aminoacid sequence of SEQ ID NO: 30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ IDNO: 53; an anti-DEspR humanized antibody comprising a variable heavy(V_(H)) chain amino acid sequence of SEQ ID NO: 55; an anti-DEspRhumanized antibody comprising a variable light (V_(L)) chain amino acidsequence of SEQ ID NO: 57; an anti-DEspR humanized antibody comprising avariable light (V_(L)) chain amino acid sequence of SEQ ID NO: 59; ananti-DEspR humanized antibody comprising a variable heavy (V_(H)) chainIgG1 amino acid sequence of SEQ ID NO: 61; an anti-DEspR humanizedantibody comprising a variable heavy (V_(H)) chain IgG4 amino acidsequence of SEQ ID NO: 63; or an anti-DEspR humanized antibodycomprising a variable light (V_(L)) chain kappa amino acid sequence ofSEQ ID NO: 65.

In some embodiments of the methods described herein, the antigen-bindingfragment is a Fab fragment. In some embodiments, the anti-DEspRantigen-binding fragment is a Fab′ fragment. In some embodiments, theanti-DEspR antigen-binding fragment is a Fd fragment. In someembodiments, the anti-DEspR antigen-binding fragment is a Fd′ fragment.In some embodiments, the antigen-binding fragment is a Fv fragment. Insome embodiments, the anti-DEspR antigen-binding fragment is a dAbfragment. In some embodiments, the anti-DEspR antigen-binding fragmentcomprises isolated CDR regions. In some embodiments, the anti-DEspRantigen-binding fragment is a F(ab′)₂ fragment. In some embodiments, theanti-DEspR antigen-binding fragment is a single chain antibody molecule.In some embodiments, the anti-DEspR antigen-binding fragment is adiabody comprising two antigen binding sites. In some embodiments, theanti-DEspR antigen-binding fragment is a linear antibody comprising apair of tandem Fd segments (V_(H)—C_(H)1-V_(H)—C_(H)1).

Accordingly, in some aspects, the disease or disorder dependent ormodulated by angiogenesis is cancer, where the rapidly dividingneoplastic cancer cells require an efficient blood supply to sustaintheir continual growth of the tumor. Inhibition of angiogenesis or tumorcell invasiveness or a combination thereof using the compositions andtherapeutic methods described herein at the primary tumor site andsecondary tumor site serve to prevent and limit metastasis andprogression of disease.

Accordingly, in some aspects, provided herein are methods to treat asubject having or at risk for a cancer or tumor comprising administeringa therapeutically effective amount of an anti-DEspR antibody orantigen-binding fragment thereof, such as a a chimeric, humanized,deimmunized, or composite human anti-DEspR antibody derived from the6G8G7 or variant 7C5B2 antibody; an anti-DEspR antibody comprising oneor more heavy chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, and SEQID NO: 23; an anti-DEspR antibody comprising one or more light chain CDRregions comprises a sequence selected from the group consisting of SEQID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43,SEQ ID NO: 44, SEQ ID NO:51, SEQ ID NO: 52, and SEQ ID NO: 53; an anti-DEspR composite humanantibody comprising a variable heavy (V_(H)) chain amino acid sequenceof SEQ ID NO: 6, SEQ ID NO: 13, or SEQ ID NO: 20; an anti-DEspRcomposite human antibody comprising a variable light (V_(L)) chain aminoacid sequence of SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 41, or SEQ IDNO: 50; an anti-DEspR antibody comprising one or more CDRs, e.g. 1 CDR,2 CDRs, 3 CDRs, 4 CDRs, 5 CDRs, or 6 CDRs, selected from the groupconsisting of (a) a heavy chain CDR1 having the amino acid sequence ofSEQ ID NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21; (b) a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 15, or SEQ IDNO: 22; (c) a heavy chain CDR3 having the amino acid sequence of SEQ IDNO: 9; SEQ ID NO: 16, or SEQ ID NO: 23; (d) a light chain CDR1 havingthe amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 35, SEQ ID NO: 42,or SEQ ID NO: 51; (e) a light chain CDR2 having the amino acid sequenceof SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 43, or SEQ ID NO: 52; and(f) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 30,SEQ ID NO: 37, SEQ ID NO: 44, or SEQ ID NO: 53; an anti-DEspR antibodycomprising a heavy chain or a fragment thereof, comprising one or moreCDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs, selected from the group consistingof a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 7,SEQ ID NO: 14, or SEQ ID NO: 21; a heavy chain CDR2 having the aminoacid sequence of SEQ ID NO: 8, SEQ ID NO: 15, or SEQ ID NO: 22; and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 9; SEQ IDNO: 16, or SEQ ID NO: 22; an anti-DEspR antibody comprising a lightchain or a fragment thereof, comprising one or more CDRs, e.g., 1 CDR, 2CDRs, or 3 CDRs selected from the group consisting of a light chain CDR1having the amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 35, SEQ IDNO: 42, or SEQ ID NO: 51; a light chain CDR2 having the amino acidsequence of SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 43, or SEQ ID NO:52; and a light chain CDR3 having the amino acid sequence of SEQ ID NO:30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ ID NO: 53; an anti-DEspRhumanized antibody comprising a variable heavy (V_(H)) chain amino acidsequence of SEQ ID NO: 55; an anti-DEspR humanized antibody comprising avariable light (V_(L)) chain amino acid sequence of SEQ ID NO: 57; ananti-DEspR humanized antibody comprising a variable light (V_(L)) chainamino acid sequence of SEQ ID NO: 59; an anti-DEspR humanized antibodycomprising a variable heavy (V_(H)) chain IgG1 amino acid sequence ofSEQ ID NO: 61; an anti-DEspR humanized antibody comprising a variableheavy (V_(H)) chain IgG4 amino acid sequence of SEQ ID NO: 63; or ananti-DEspR humanized antibody comprising a variable light (V_(L)) chainkappa amino acid sequence of SEQ ID NO: 65.

In some embodiments of the methods described herein, the methods canfurther comprise first selecting or diagnosing the subject having or atrisk for a cancer or tumor. In some such embodiments, the diagnosis ofthe subject can comprise administering to the subject an anti-DEspRantibody or antigen-binding fragment thereof coupled to a label, forexample, a radioactive label, or a label used for molecular imaging, asdescribed elsewhere herein. In such embodiments, detection of thelabeled anti-DEspR antibody or antigen-binding fragment is indicative ofthe subject having a cancer or tumor.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Accordingly, the terms “cancer” or “tumor” asused herein refers to an uncontrolled growth of cells which interfereswith the normal functioning of the bodily organs and systems, includingcancer stem cells and tumor vascular niches. A subject that has a canceror a tumor is a subject having objectively measurable cancer cellspresent in the subject's body. Cancers which migrate from their originallocation and seed vital organs can eventually lead to the death of thesubject through the functional deterioration of the affected organs.Hematopoietic cancers, such as leukemia, are able to out-compete thenormal hematopoietic compartments in a subject, thereby leading tohematopoietic failure (in the form of anemia, thrombocytopenia andneutropenia) ultimately causing death.

By “metastasis” is meant the spread of cancer from its primary site toother places in the body. Cancer cells can break away from a primarytumor, penetrate into lymphatic and blood vessels, circulate through thebloodstream, and grow in a distant focus (metastasize) in normal tissueselsewhere in the body. Metastasis can be local or distant. Metastasis isa sequential process, contingent on tumor cells breaking off from theprimary tumor, traveling through the bloodstream, and stopping at adistant site. At the new site, the cells establish a blood supply andcan grow to form a life-threatening mass. Both stimulatory andinhibitory molecular pathways within the tumor cell regulate thisbehavior, and interactions between the tumor cell and host cells in thedistant site are also significant.

Metastases are most often detected through the sole or combined use ofmagnetic resonance imaging (MRI) scans, computed tomography (CT) scans,blood and platelet counts, liver function studies, chest X-rays and bonescans in addition to the monitoring of specific symptoms.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include, but are not limited to, basal cell carcinoma, biliarytract cancer; bladder cancer; bone cancer; brain and CNS cancer; breastcancer; cancer of the peritoneum; cervical cancer; choriocarcinoma;colon and rectum cancer; connective tissue cancer; cancer of thedigestive system; endometrial cancer; esophageal cancer; eye cancer;cancer of the head and neck; gastric cancer (including gastrointestinalcancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelialneoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer;lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; glioblastoma; oral cavity cancer (e.g., lip,tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostatecancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; as well as other carcinomas and sarcomas; as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

In other aspects, the methods described herein are used in the treatmentor inhibition or imaging of artherosclerotic plaques andatherosclerosis. The process of atherosclerosis involves inflammation,and white blood cells (e.g., lymphocytes, monocytes, and macrophages)are often present throughout the development of atherosclerosis.Atherosclerosis begins when monocytes are activated and move out of thebloodstream into the wall of an artery. There, they are transformed intofoam cells, which collect cholesterol and other fatty materials. Intime, these fat-laden foam cells accumulate and form atheromas in thelining of the artery's wall, causing a thickening and hardening of thewall. Atheromas can be scattered throughout medium-sized and largearteries, but usually form where the arteries branch. Treatment of anddiagnosis of atherosclerosis is important because it often leads toheart disease and can also cause stroke or other vascular problems suchas claudication.

Accordingly, in some embodiments of the aspects described herein,pathological angiogenesis in atherosclerotic plaques and in the vasavasorum of atherosclerotic arteries (coronary and carotid arterydisease) is considered a risk and/or causal factor for vulnerable plaqueprogression and disruption. Thus, in some such embodiments, a subjecthaving an angiogenic disorder to be treated using the compositions andmethods described herein can have or be at risk for atherosclerosis. Asused herein, “atherosclerosis” refers to a disease of the arterial bloodvessels resulting in the hardening of arteries caused by the formationof multiple atheromatous plaques within the arteries. Atherosclerosiscan be associated with other disease conditions, including but notlimited to, coronary heart disease events, cerebrovascular events, acutecoronary syndrome, and intermittent claudication. For example,atherosclerosis of the coronary arteries commonly causes coronary arterydisease, myocardial infarction, coronary thrombosis, and anginapectoris. Atherosclerosis of the arteries supplying the central nervoussystem frequently provokes strokes and transient cerebral ischemia. Inthe peripheral circulation, atherosclerosis causes intermittentclaudication and gangrene and can jeopardize limb viability.Atherosclerosis of an artery of the splanchnic circulation can causemesenteric ischemia. Atherosclerosis can also affect the kidneysdirectly (e.g., renal artery stenosis). Also, persons who havepreviously experienced one or more non-fatal atherosclerotic diseaseevents are those for whom the potential for recurrence of such an eventexists.

Sometimes these other diseases can be caused by or associated with otherthan atherosclerosis. Therefore, in some embodiments, one firstdiagnoses that atherosclerosis is present prior to administering thecompositions described herein to the subject. A subject is “diagnosedwith atherosclerosis” or “selected as having atherosclerosis” if atleast one of the markers of symptoms of atherosclerosis is present. Inone such embodiment, the subject is “selected” if the person has afamily history of atherosclerosis or carries a known genetic mutation orpolymorphism for high cholesterol. In one embodiment, a subject isdiagnosed by measuring an increase level of C-reactive protein (CRP) inthe absence of other inflammatory disorders. In other embodiments,atherosclerosis is diagnosed by measuring serum levels of homocysteine,fibrinogen, lipoprotein (a), or small LDL particles. Alternatively acomputed tomography scan, which measures calcium levels in the coronaryarteries, can be used to select a subject having atherosclerosis. In oneembodiment, atherosclerosis is diagnosed by an increase in inflammatorycytokines. In one embodiment, increased interleukin-6 levels are used asan indicator to select an individual having atherosclerosis. In otherembodiments, increased interleukin-8 and/or interleukin-17 level is usedas an indicator to select an individual having atherosclerosis.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in age-relatedmacular degeneration. It is known, for example, that VEGF contributes toabnormal blood vessel growth from the choroid layer of the eye into theretina, similar to what occurs during the wet or neovascular form ofage-related macular degeneration. Macular degeneration, often called AMDor ARMD (age-related macular degeneration), is the leading cause ofvision loss and blindness in Americans aged 65 and older. New bloodvessels grow (neovascularization) beneath the retina and leak blood andfluid. This leakage causes permanent damage to light-sensitive retinalcells, which die off and create blind spots in central vision or themacula. Accordingly, encompassed in the methods disclosed herein aresubjects treated for age-related macular degeneration withanti-angiogenic therapy.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in a subject havingdiabetic retinopathy, where abnormal blood vessel growth is associatedwith diabetic eye diseases and diabetic macular edema. When normal bloodvessels in the retina are damaged by tiny blood clots due to diabetes, achain reaction is ignited that culminates in new blood vessel growth.However, the backup blood vessels are faulty; they leak (causing edema),bleed and encourage scar tissue that detaches the retina, resulting insevere loss of vision. Such growth is the hallmark of diabeticretinopathy, the leading cause of blindness among young people indeveloped countries. Therefore, encompassed in the methods disclosedherein are subjects treated for diabetic retinopathy and/or diabeticmacular edema.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in a subject havingrheumatoid arthritis. Rheumatoid arthritis (RA) is characterized bysynovial tissue swelling, leukocyte ingress and angiogenesis, or newblood vessel growth. The expansion of the synovial lining of joints inrheumatoid arthritis (RA) and the subsequent invasion by the pannus ofunderlying cartilage and bone necessitate an increase in the vascularsupply to the synovium, to cope with the increased requirement foroxygen and nutrients. Angiogenesis is now recognized as a key event inthe formation and maintenance of the pannus in RA (Paleolog, E. M.,Arthritis Res. 2002;4 Suppl 3:S81-90; Afuwape A O, Histol Histopathol.2002;17(3):961-72). Even in early RA, some of the earliest histologicalobservations are blood vessels. A mononuclear infiltrate characterizesthe synovial tissue along with a luxuriant vasculature. Angiogenesis isintegral to formation of the inflammatory pannus and withoutangiogenesis, leukocyte ingress could not occur (Koch, A. E., Ann.Rheum. Dis. 2000, 59 Suppl 1:i65-71). Disruption of the formation of newblood vessels would not only prevent delivery of nutrients to theinflammatory site, it could also reduce joint swelling due to theadditional activity of VEGF, a potent proangiogenic factor in RA, as avascular permeability factor. Anti-VEGF hexapeptide RRKRRR (dRK6) cansuppress and mitigate the arthritis severity (Seung-Ah Yoo, et.al.,2005, supra). Accordingly, encompassed in the methods disclosedherein are subjects having or being treated for rheumatoid arthritis.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in Alzheimer'sdisease. Alzheimer's disease (AD) is the most common cause of dementiaworldwide. AD is characterized by an excessive cerebral amyloiddeposition leading to degeneration of neurons and eventually todementia. The exact cause of AD is still unknown. It has been shown byepidemiological studies that long-term use of non-steroidalanti-inflammatory drugs, statins, histamine H2-receptor blockers, orcalcium-channel blockers, all of which are cardiovascular drugs havinganti-angiogenic effects, seem to prevent Alzheimer's disease and/orinfluence the outcome of AD patients. Therefore, AD angiogenesis in thebrain vasculature can play an important role in AD. In Alzheimer'sdisease, the brain endothelium secretes the precursor substrate for thebeta-amyloid plaque and a neurotoxic peptide that selectively killscortical neurons. Moreover, amyloid deposition in the vasculature leadsto endothelial cell apoptosis and endothelial cell activation whichleads to neovascularization. Vessel formation could be blocked by theVEGF antagonist SU 4312 as well as by statins, indicating thatanti-angiogenesis strategies can interfere with endothelial cellactivation in AD (Schultheiss C., el. al., 2006; Grammas P., et. al.,1999) and can be used for preventing and/or treating AD. Accordingly,encompassed in the methods disclosed herein are subjects being treatedfor Alzheimer's disease.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in ischemic regionsin the brain, which can contribute to edema, leaky neovessels, andpredispose a subject to hemorrhagic transformation after an ischemicstroke event, thus worsening the morbidity and mortality risk from thestroke event. Inhibition of leaky angiogenic neovessels using thecompositions and methods described herein can reduce neurologic deficitsfrom an ischemic stroke event, as well as prevent the progression tohemorrhagic stroke. Currently, there is no therapy for ischemichemorrhagic transformation nor effective therapies to reduce theneurologic deficits from stroke.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in obesity.Adipogenesis in obesity involves interplay between differentiatingadipocytes, stromal cells, and blood vessels. Close spatial and temporalinterrelationships between blood vessel formation and adipogenesis, andthe sprouting of new blood vessels from preexisting vasculature wascoupled to adipocyte differentiation. Adipogenic/angiogenic cellclusters can morphologically and immunohistochemically be distinguishedfrom crown-like structures frequently seen in the late stages of adiposetissue obesity. Administration of anti-vascular endothelial growthfactor (VEGF) antibodies inhibited not only angiogenesis but also theformation of adipogenic/angiogenic cell clusters, indicating that thecoupling of adipogenesis and angiogenesis is essential fordifferentiation of adipocytes in obesity and that VEGF is a key mediatorof that process. (Satoshi Nishimura et. al., 2007, Diabetes56:1517-1526). It has been shown that the angiogenesis inhibitor,TNP-470 was able to prevent diet-induced and genetic obesity in mice(Ebba Bråkenhielm et. al., Circulation Research, 2004;94:1579). TNP-470reduced vascularity in the adipose tissue, thereby inhibiting the rateof growth of the adipose tissue and obesity development. Accordingly,encompassed in the methods disclosed herein are subjects suffering fromobesity.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in endometriosis.Excessive endometrial angiogenesis is proposed as an important mechanismin the pathogenesis of endometriosis (Healy, DL., et. al., Hum ReprodUpdate. 1998 Sep-Oct;4(5):736-40). The endometrium of patients withendometriosis shows enhanced endothelial cell proliferation. Moreoverthere is an elevated expression of the cell adhesion molecule integrinvβ3 in more blood vessels in the endometrium of women with endometriosiswhen compared with normal women. The U.S. Pat. No. 6,121,230 describedthe use of anti-VEGF agents in the treatment of endometriosis and isPatent is incorporated hereby reference. Accordingly, encompassed in themethods disclosed herein are subjects having or being treated forendometriosis.

As described herein, any of a variety of tissues, or organs comprised oforganized tissues, can support angiogenesis in disease conditionsincluding skin, muscle, gut, connective tissue, joints, bones and thelike tissue in which blood vessels can invade upon angiogenic stimuli.

The individual or subject to be treated as described herein in variousembodiments is desirably a human patient, although it is to beunderstood that the methods are effective with respect to all mammals,which are intended to be included in the term “patient” or “subject”. Inthis context, a mammal is understood to include any mammalian species inwhich treatment of diseases associated with angiogenesis is desirable.The terms “subject” and “individual” are used interchangeably herein,and refer to an animal, for example a human, recipient of theDEspR-specific antibodies and antigen-binding fragments describedherein. For treatment of disease states which are specific for aspecific animal such as a human subject, the term “subject” refers tothat specific animal. The terms “non-human animals” and “non-humanmammals” are used interchangeably herein, and include mammals such asrats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-humanprimates. The term “subject” also encompasses any vertebrate includingbut not limited to mammals, reptiles, amphibians and fish. However,advantageously, the subject is a mammal such as a human, or othermammals such as a domesticated mammal, e.g. dog, cat, horse, and thelike, or production mammal, e.g. cow, sheep, pig, and the like are alsoencompassed in the term subject.

The DEspR-specific antagonist agents, such as anti-DEspR antibodies orantigen-binding fragments thereof, described herein can be administeredto a subject in need thereof by any appropriate route which results inan effective treatment in the subject. As used herein, the terms“administering,” and “introducing” are used interchangeably and refer tothe placement of an anti-DEspR antibody or antigen-binding fragmentthereof into a subject by a method or route which results in at leastpartial localization of such agents at a desired site, such as a site ofinflammation or cancer, such that a desired effect(s) is produced.

In some embodiments, the anti-DEspR antibody or antigen-binding fragmentthereof is administered to a subject having an angiogenic disorder to beinhibited by any mode of administration that delivers the agentsystemically or to a desired surface or target, and can include, but isnot limited to, injection, infusion, instillation, and inhalationadministration. To the extent that anti-DEspR antibodies orantigen-binding fragments thereof can be protected from inactivation inthe gut, oral administration forms are also contemplated. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments, the anti-DEspR antibodies or antigen-bindingfragments thereof for use in the methods described herein areadministered by intravenous infusion or injection.

The phrases “parenteral administration” and “administered parenterally”as used herein, refer to modes of administration other than enteral andtopical administration, usually by injection. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein refer tothe administration of the bispecific or multispecific polypeptide agentother than directly into a target site, tissue, or organ, such as atumor site, such that it enters the subject's circulatory system and,thus, is subject to metabolism and other like processes.

The anti-DEspR antibodies or antigen-binding fragments thereof describedherein are administered to a subject, e.g., a human subject, in accordwith known methods, such as intravenous administration as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes. In someembodiments of the methods described herein, local administration, forexample, to a tumor or cancer site where angiogenesis is occurring, canbe used to increase effectiveness of the anti-DEspR antibody orantigen-binding fragment thereof and/or to help reduce side effects ortoxicity.

In some embodiments, the anti-DEspR antibody or antigen-binding fragmentthereof is administered by any suitable means, including parenteral,subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, ifdesired for local immunosuppressive treatment, intralesionaladministration. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration. In someembodiments, the antibody or antigen-binding fragment thereof issuitably administered by pulse infusion, particularly with decliningdoses of the antibody. Preferably the dosing is given by injections,most preferably intravenous or subcutaneous injections, depending inpart on whether the administration is brief or chronic.

In some embodiments, the anti-DEspR antibody or antigen-binding fragmentthereof is administered locally, e.g., by direct injections, when thedisorder or location of the tumor permits, and the injections can berepeated periodically. The DEspR-specific antagonist can also bedelivered systemically to the subject or directly to the tumor cells,e.g., to a tumor or a tumor bed following surgical excision of thetumor, in order to prevent or reduce local recurrence or metastasis, forexample of a dormant tumor or micrometastases.

Antibody-targeted sonoporation methods are contemplated for use in someembodiments of the methods for inhibiting angiogenesis described herein,in order to enhance the efficacy and potency of the therapeuticcompositions comprising anti-DEspR antibodies and antigen-bindingfragments thereof provided herein. Accordingly, in some embodiments ofthe methods of inhibiting angiogenesis described herein, anti-DEspRantibodies and antigen-binding fragments thereof are administered to asubject in need thereof by sonoporation.

As used herein, “sonoporation” refers to the use of sound, preferably atultrasonic frequencies, or the interaction of ultrasound with contrastagents (e.g., stabilized microbubbles) for temporarily modifying thepermeability of cell plasma membranes, thus allowing uptake of largemolecules, such as therapeutic agents. The membrane permeability causedby the sonoporation is transient, leaving the agents trapped inside thecell after the ultrasound exposure. Sonoporation employs acousticcavitation of microbubbles to enhance delivery of large molecules.

Accordingly, in some embodiments of the methods, the anti-DEspRantibodies and antigen-binding fragments thereof described herein, mixedwith ultrasound contrast agents, such as microbubbles, can be injectedlocally or systemically into a subject in need of treatment for anangiogenic disorder, and ultrasound can be coupled and even focused intothe defined area, e.g., tumor site, to achieve targeted delivery of theanti-DEspR antibodies and antigen-binding fragments thereof describedherein. In addition to the operator-determined focused ultrasound,anti-DEspR targeting of a microbubble can be used to target thesonoporation-mediated enhanced entry of any therapeutic agent, includingantiDEspR monoclonal antibody per se, into said targeted cancerousareas.

In some embodiments, the methods use focused ultrasound methods toachieve targeted delivery of the anti-DEspR antibodies andantigen-binding fragments thereof described herein. As used herein, HIFUor “High Intensity Focused Ultrasound” refers to a non-invasivetherapeutic method using high-intensity ultrasound to heat and destroymalignant or pathogenic tissue without causing damage to overlying orsurrounding health tissue. Typically, HIFU has been used in tissueablation techniques, whereby the biological effects of HIFU treatment,including coagulative necrosis and structural disruption, can be inducedin a tissue requiring ablation, such as a solid tumor site. However, asdescribed in Khaibullina A. et al., J Nucl Med. 2008 Feb;49(2):295-302,and WO2010127369, the contents of which are herein incorporated in theirentireties by reference, HIFU can also be used as a means of delivery oftherapeutic agents, such as antibodies or antigen-binding fragmentsthereof.

Methods using contrast-enhanced ultrasound (CEUS) are also contemplatedfor use with anti-DEspR inhibiting agents described herein.Contrast-enhanced ultrasound (CEUS) refers to the application ofultrasound contrast medium and ultrasound contrast agents to traditionalmedical sonography. Ultrasound contrast agents refer to agents that relyon the different ways in which sound waves are reflected from interfacesbetween substances. This can be the surface of a small air bubble or amore complex structure. Commercially available contrast media includegas-filled microbubbles that are administered intravenously to thesystemic circulation. Microbubbles have a high degree of echogenicity,i.e., the ability of an object to reflect the ultrasound waves. Theechogenicity difference between the gas in the microbubbles and the softtissue surroundings of the body is immense, and enhances the ultrasoundbackscatter, or reflection of the ultrasound waves, to produce a uniquesonogram with increased contrast due to the high echogenicitydifference. Contrast-enhanced ultrasound can be used with thecompositions and methods described herein to image a variety ofconditions and disorders, such as angiogenesis dependent disorders, asdescribed herein

A variety of microbubble contrast agents are available for use with thecompositions and methods described herein. Microbubbles can differ intheir shell makeup, gas core makeup, and whether or not they aretargeted.

The microbubble shell material determines how easily the microbubble istaken up by the immune system. A more hydrophilic shell material tendsto be taken up more easily, which reduces the microbubble residence timein the circulation. This reduces the time available for contrastimaging. The shell material also affects microbubble mechanicalelasticity. The more elastic the material, the more acoustic energy itcan withstand before bursting. Example of materials used in currentmicrobubble shells include albumin, galactose, lipid, and polymers, asdescribed in Lindner, J.R. 2004. Microbubbles in medical imaging:current applications and future directions. Nat Rev Drug Discov. 3:527-32, the contents of which are herein incoporated by reference intheir entireties.

The microbubble gas core is an important part of the ultrasound contrastmicrobubble because it determines the echogenicity. When gas bubbles arecaught in an ultrasonic frequency field, they compress, oscillate, andreflect a characteristic echo—this generates the strong and uniquesonogram in contrast-enhanced ultrasound. Gas cores can be composed of,for example, air, or heavy gases like perfluorocarbon, or nitrogen.Heavy gases are less water-soluble so they are less likely to leak outfrom the microbubble to impair echogenicity. Therefore, microbubbleswith heavy gas cores are likely to last longer in circulation.

Regardless of the shell or gas core composition, microbubble size aretypically fairly uniform. They can lie within in a range of 1-4micrometres in diameter. That makes them smaller than red blood cells,which allows them to flow easily through the circulation as well as themicrocirculation.

Targeting ligands that bind to receptors characteristic of angiogenicdisorders, such as DEspR, can be conjugated to microbubbles, enablingthe microbubble complex to accumulate selectively in areas of interest,such as diseased or abnormal tissues. This form of molecular imaging,known as targeted contrast-enhanced ultrasound, will only generate astrong ultrasound signal if targeted microbubbles bind in the area ofinterest. Targeted contrast-enhanced ultrasound has many applications inboth medical diagnostics and medical therapeutics. Microbubbles targetedwith an anti-DEspR antibody or antigen-binding fragment thereof areinjected systemically in a small bolus. These DEspR-targetedmicrobubbles can travel through the circulatory system, eventuallyfinding their respective targets and binding specifically. Ultrasoundwaves can then be directed on the area of interest. If a sufficientnumber of DEspR-targeted microbubbles have bound in the area, theircompressible gas cores oscillate in response to the high frequency sonicenergy field. The DEspR-targeted microbubbles also reflect a unique echothat is in stark contrast to the surrounding tissue due to the orders ofmagnitude mismatch between microbubble and tissue echogenicity. Theultrasound system converts the strong echogenicity into acontrast-enhanced image of the area of interest, revealing the locationof the bound DEspR-targeted microbubbles. Detection of boundmicrobubbles can then show that the area of interest is expressingDEspR, which can be indicative of a certain disease state, or identifyparticular cells in the area of interest. In addition, targetedsonoporation can be done at the site where DEspR-targeted microbubblesare attached, thus achieving targeted delivery of any therapeutic agent(drug, siRNA, DNA, small molecule) encapsulated in or carried on theechogenic microbubble.

Accordingly, in some embodiments of the methods described herein, ananti-DEspR antibody or antigen-binding fragment thereof, such as achimeric, humanized, deimmunized, or composite human anti-DEspR antibodyderived from the 6G8G7 or variant 7C5B2 antibody; an anti-DEspR antibodycomprising one or more heavy chain CDR regions comprising a sequenceselected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQID NO: 22, and SEQ ID NO: 23; an anti-DEspR antibody comprising one ormore light chain CDR regions comprises a sequence selected from thegroup consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43,SEQID NO: 44, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53; ananti-DEspR composite human antibody comprising a variable heavy (V_(H))chain amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 13, or SEQ ID NO:20; an anti-DEspR composite human antibody comprising a variable light(V_(L)) chain amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 34, SEQID NO: 41, or SEQ ID NO: 50; an anti-DEspR antibody comprising one ormore CDRs, e.g. 1 CDR, 2 CDRs, 3 CDRs, 4 CDRs, 5 CDRs, or 6 CDRs,selected from the group consisting of (a) a heavy chain CDR1 having theamino acid sequence of SEQ ID NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21;(b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8,SEQ ID NO: 15, or SEQ ID NO: 22; (c) a heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 9; SEQ ID NO: 16, or SEQ ID NO: 23; (d) alight chain CDR1 having the amino acid sequence of SEQ ID NO: 28, SEQ IDNO: 35, SEQ ID NO: 42, or SEQ ID NO: 51; (e) a light chain CDR2 havingthe amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 43,or SEQ ID NO: 52; and (f) a light chain CDR3 having the amino acidsequence of SEQ ID NO: 30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ ID NO:53; an anti-DEspR antibody comprising a heavy chain or a fragmentthereof, comprising one or more CDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs,selected from the group consisting of a heavy chain CDR1 having theamino acid sequence of SEQ ID NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21; aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8, SEQ IDNO: 15, or SEQ ID NO: 22; and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 9; SEQ ID NO: 16, or SEQ ID NO: 22; an anti-DEspRantibody comprising a light chain or a fragment thereof, comprising oneor more CDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs selected from the groupconsisting of a light chain CDR1 having the amino acid sequence of SEQID NO: 28, SEQ ID NO: 35, SEQ ID NO: 42, or SEQ ID NO: 51; a light chainCDR2 having the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 36, SEQID NO: 43, or SEQ ID NO: 52; and a light chain CDR3 having the aminoacid sequence of SEQ ID NO: 30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ IDNO: 53; an anti-DEspR humanized antibody comprising a variable heavy(V_(H)) chain amino acid sequence of SEQ ID NO: 55; an anti-DEspRhumanized antibody comprising a variable light (V_(L)) chain amino acidsequence of SEQ ID NO: 57; an anti-DEspR humanized antibody comprising avariable light (V_(L)) chain amino acid sequence of SEQ ID NO: 59; ananti-DEspR humanized antibody comprising a variable heavy (V_(H)) chainIgG1 amino acid sequence of SEQ ID NO: 61; an anti-DEspR humanizedantibody comprising a variable heavy (V_(H)) chain IgG4 amino acidsequence of SEQ ID NO: 63; or an anti-DEspR humanized antibodycomprising a variable light (V_(L)) chain kappa amino acid sequence ofSEQ ID NO: 65, is administered to a subject in need of treatment for anangiogenic disorder, such as for example, cancer, using a targetedultrasound delivery. In some such embodiments, the targeted ultrasounddelivery comprises using microbubbles as contrast agents to which ananti-DEspR antibody or antigen-binding fragment thereof. In some suchembodiments, the targeted ultrasound is HIFU.

For the clinical use of the methods described herein, administration ofthe anti-DEspR antibodies or antigen-binding fragments thereof describedherein, can include formulation into pharmaceutical compositions orpharmaceutical formulations for parenteral administration, e.g.,intravenous; mucosal, e.g., intranasal; ocular, or other mode ofadministration. In some embodiments, the anti-DEspR antibodies orantigen-binding fragments thereof described herein can be administeredalong with any pharmaceutically acceptable carrier compound, material,or composition which results in an effective treatment in the subject.Thus, a pharmaceutical formulation for use in the methods describedherein can contain an anti-DEspR antibody or antigen-binding fragmentthereof as described herein in combination with one or morepharmaceutically acceptable ingredients.

The phrase “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio. The phrase “pharmaceutically acceptablecarrier” as used herein means a pharmaceutically acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent, media, encapsulating material, manufacturing aid(e.g., lubricant, talc magnesium, calcium or zinc stearate, or stericacid), or solvent encapsulating material, involved in maintaining thestability, solubility, or activity of, an anti-DEspR antibody orantigen-binding fragment thereof. Each carrier must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not injurious to the patient. The terms “excipient”,“carrier”, “pharmaceutically acceptable carrier” or the like are usedinterchangeably herein.

The anti-DEspR antibodies or antigen-binding fragments thereof describedherein can be specially formulated for administration of the compound toa subject in solid, liquid or gel form, including those adapted for thefollowing: (1) parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation; (2)topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin; (3)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (4) ocularly; (5) transdermally; (6) transmucosally; or (79)nasally. Additionally, an anti-DEspR antibody or antigen-bindingfragment thereof can be implanted into a patient or injected using adrug delivery system. See, for example, Urquhart, et al., Ann. Rev.Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Releaseof Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S.Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960.

Therapeutic formulations of the anti-DEspR antibodies or antigen-bindingfragments thereof described herein can be prepared for storage by mixingthe anti-DEspR antibodies or antigen-binding fragments having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Exemplary lyophilized anti-VEGF antibodyformulations are described in WO 97/04801, expressly incorporated hereinbe reference.

Optionally, but preferably, the formulations comprising the compositionsdescribed herein contain a pharmaceutically acceptable salt, typically,e.g., sodium chloride, and preferably at about physiologicalconcentrations. Optionally, the formulations of the invention cancontain a pharmaceutically acceptable preservative. In some embodimentsthe preservative concentration ranges from 0.1 to 2.0%, typically v/v.Suitable preservatives include those known in the pharmaceutical arts.Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben areexamples of preservatives. Optionally, the formulations of the inventioncan include a pharmaceutically acceptable surfactant at a concentrationof 0.005 to 0.02%.

The therapeutic formulations of the compositions comprising anti-DEspRantibodies and antigen-binding fragments thereofdescribed herein canalso contain more than one active compound as necessary for theparticular indication being treated, preferably those with complementaryactivities that do not adversely affect each other. For example, in someembodiments, it can be desirable to further provide antibodies whichbind to EGFR, VEGF (e.g. an antibody which binds a different epitope onVEGF), VEGFR, or ErbB2 (e.g., Herceptin™). Alternatively, or inaddition, the composition can comprise a cytotoxic agent, cytokine,growth inhibitory agent, and/or VEGFR antagonist. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended.

The active ingredients of the therapeutic formulations of thecompositions comprising the anti-DEspR antibodies or antigen-bindingfragments described herein can also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

In some embodiments, sustained-release preparations can be used.Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theanti-DEspR antibodies or antigen-binding fragments in which the matricesare in the form of shaped articles, e.g., films, or microcapsule.Examples of sustained-release matrices include polyesters, hydrogels(for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they can denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization can be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

The therapeutic formulations to be used for in vivo administration, suchas parenteral administration, in the methods described herein can besterile, which is readily accomplished by filtration through sterilefiltration membranes, or other methods known to those of skill in theart.

The anti-DEspR antibodies and antigen-binding fragments thereof, areformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular subject being treated,the clinical condition of the individual subject, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the anti-DEspR antibodies and antigen-binding fragments thereof to beadministered will be governed by such considerations, and refers to theminimum amount necessary to ameliorate, treat, or stabilize, the cancer;to increase the time until progression (duration of progression freesurvival) or to treat or prevent the occurrence or recurrence of atumor, a dormant tumor, or a micrometastases. The anti-DEspR antibodiesand antigen-binding fragments thereof are optionally formulated with oneor more additional therapeutic agents currently used to prevent or treatcancer or a risk of developing a cancer. The effective amount of suchother agents depends on the amount of anti-DEspR antibodies andantigen-binding fragments thereof present in the formulation, the typeof disorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused herein before or about from 1 to 99% of the heretofore employeddosages.

The dosage ranges for the agent depend upon the potency, and encompassamounts large enough to produce the desired effect e.g., slowing oftumor growth or a reduction in tumor size. The dosage should not be solarge as to cause unacceptable adverse side effects. Generally, thedosage will vary with the age, condition, and sex of the patient and canbe determined by one of skill in the art. The dosage can also beadjusted by the individual physician in the event of any complication.In some embodiments, the dosage ranges from 0.001 mg/kg body weight to100 mg/kg body weight. In some embodiments, the dose range is from 5μg/kg body weight to 100 μg/kg body weight. Alternatively, the doserange can be titrated to maintain serum levels between 1 μg/mL and 1000μg/mL. For systemic administration, subjects can be administered atherapeutic amount, such as, e.g., 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more. These doses can beadministered by one or more separate administrations, or by continuousinfusion. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until, forexample, the cancer is treated, as measured by the methods describedabove or known in the art. However, other dosage regimens can be useful.

In some embodiments, the anti-DEspR antibody or antigen-binding fragmentthereof is administered once every week, every two weeks, or every threeweeks, at a dose range from about 2 mg/kg to about 15 mg/kg, including,but not limited to 5 mg/kg, 7.5 mg/kg, 10 mg/kg or 15 mg/kg. Theprogress of using the methods described herein can be easily monitoredby conventional techniques and assays. In some embodiments the dose canbe administered about weekly. In some embodiments, the dose can beadministered weekly. In some embodiments, the dose can be administeredweekly for from about 12 weeks to about 18 weeks. In some embodimentsthe dose can be administered about every 2 weeks. In some embodimentsthe dose can be administered about every 3 weeks. In some embodiments,the dose can be from about 2 mg/kg to about 15 mg/kg administered aboutevery 2 weeks. In some embodiments, the dose can be from about 2 mg/kgto about 15 mg/kg administered about every 3 weeks. In some embodiments,the dose can be from about 2 mg/kg to about 15 mg/kg administeredintravenously about every 2 weeks. In some embodiments, the dose can befrom about 2 mg/kg to about 15 mg/kg administered intravenously aboutevery 3 weeks. In some embodiments, a total of from about 2 to about 10doses are administered. In some embodiments, a total of 4 doses areadministered. In some embodiments, a total of 5 doses are administered.In some embodiments, a total of 6 doses are administered. In someembodiments, a total of 7 doses are administered. In some embodiments, atotal of 8 doses are administered. In some embodiments, theadministration occurs for a total of from about 4 weeks to about 12weeks. In some embodiments, the administration occurs for a total ofabout 6 weeks. In some embodiments, the administration occurs for atotal of about 8 weeks. In some embodiments, the administration occursfor a total of about 12 weeks. In some embodiments, the initial dose canbe from about 1.5 to about 2.5 fold greater than subsequent doses.

The duration of a therapy using the methods described herein willcontinue for as long as medically indicated or until a desiredtherapeutic effect (e.g., those described herein) is achieved. Incertain embodiments, the administration of the DEspR-specific antibodyor antigen-binding fragment described herein is continued for 1 month, 2months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3years, 4 years, 5 years, 10 years, 20 years, or for a period of years upto the lifetime of the subject.

The efficacy of the treatment methods for cancer comprising therapeuticformulations of the compositions comprising anti-DEspR antibodies orantigen-binding fragments thereof described herein can be measured byvarious endpoints commonly used in evaluating cancer treatments,including but not limited to, tumor regression, tumor weight or sizeshrinkage, time to progression, duration of survival, progression freesurvival, overall response rate, duration of response, and quality oflife. Because the anti-DEspR antibodies and antigen-binding fragmentsthereof described herein target the tumor vasculature, cancer cells, andsome cancer stem cell subsets, they represent a unique class ofmulti-targeting anticancer drugs, and therefore can require uniquemeasures and definitions of clinical responses to drugs. For example,tumor shrinkage of greater than 50% in a 2-dimensional analysis is thestandard cut-off for declaring a response. However, theanti-DEspR-antibodies or antigen-binding fragments thereof describedherein can cause inhibition of metastatic spread without shrinkage ofthe primary tumor, or can simply exert a tumoristatic effect.Accordingly, novel approaches to determining efficacy of ananti-angiogenic therapy should be employed, including for example,measurement of plasma or urinary markers of angiogenesis, andmeasurement of response through molecular imaging, using, for example,an DEspR-antibody or antigen-binding fragment conjugated to a label,such as a microbubble. In the case of cancers, the therapeuticallyeffective amount of the DEspR-antibody or antigen-binding fragmentthereof can reduce the number of cancer cells; reduce the tumor size;inhibit (i.e., slow to some extent and preferably stop) cancer cellinfiltration into peripheral organs; inhibit (i.e., slow to some extentand preferably stop) tumor metastasis; inhibit, to some extent, tumorgrowth; and/or relieve to some extent one or more of the symptomsassociated with the disorder. To the extent the DEspR-antibody orantigen-binding fragment thereof can prevent growth and/or kill existingcancer cells, it can be cytostatic and/or cytotoxic. For cancer therapy,efficacy in vivo can, for example, be measured by assessing the durationof survival, duration of progression free survival (PFS), the responserates (RR), duration of response, and/or quality of life.

In other embodiments, described herein are methods for increasingprogression free survival of a human subject susceptible to or diagnosedwith a cancer. Time to disease progression is defined as the time fromadministration of the drug until disease progression or death. In apreferred embodiment, the combination treatment using an anti-DEspRantibody or antigen-binding fragment thereof, and one or morechemotherapeutic agents significantly increases progression freesurvival by at least about 1 month, 1.2 months, 2 months, 2.4 months,2.9 months, 3.5 months, preferably by about 1 to about 5 months, whencompared to a treatment with chemotherapy alone. In another embodiment,the methods decribed herein significantly increase response rates in agroup of human subjects susceptible to or diagnosed with a cancer whoare treated with various therapeutics. Response rate is defined as thepercentage of treated subjects who responded to the treatment. In oneembodiment, the combination treatment described herein using aDEspR-specific antagonist, such as an anti-DEspR antibody orantigen-binding fragment thereof, and one or more chemotherapeuticagents significantly increases response rate in the treated subjectgroup compared to the group treated with chemotherapy alone.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with, a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorderassociated with a chronic immune condition, such as, but not limited to,a chronic infection or a cancer. Treatment is generally “effective” ifone or more symptoms or clinical markers are reduced. Alternatively,treatment is “effective” if the progression of a disease is reduced orhalted. That is, “treatment” includes not just the improvement ofsymptoms or markers, but also a cessation of at least slowing ofprogress or worsening of symptoms that would be expected in absence oftreatment. Beneficial or desired clinical results include, but are notlimited to, alleviation of one or more symptom(s), diminishment ofextent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. The term “treatment” of a disease alsoincludes providing relief from the symptoms or side-effects of thedisease (including palliative treatment).

For example, in some embodiments, the methods described herein compriseadministering an effective amount of the anti-DEspR antibodies orantigen-binding fragments thereof described herein to a subject in orderto alleviate a symptom of a cancer, or other such disorder characterizedby excess or unwanted angiogenesis. As used herein, “alleviating asymptom of a cancer” is ameliorating or reducing any condition orsymptom associated with the cancer. As compared with an equivalentuntreated control, such reduction or degree of prevention is at least5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by anystandard technique. Ideally, the cancer is completely cleared asdetected by any standard method known in the art, in which case thecancer is considered to have been treated. A patient who is beingtreated for a cancer is one who a medical practitioner has diagnosed ashaving such a condition. Diagnosis can be by any suitable means.Diagnosis and monitoring can involve, for example, detecting the levelof cancer cells in a biological sample (for example, a tissue or lymphnode biopsy, blood test, or urine test), detecting the level of asurrogate marker of the cancer in a biological sample, detectingsymptoms associated with the specific cancer, or detecting immune cellsinvolved in the immune response typical of such a cancer.

The term “effective amount” as used herein refers to the amount of ananti-DEspR antibody or antigen-binding fragment thereof needed toalleviate at least one or more symptom of the disease or disorder, andrelates to a sufficient amount of pharmacological composition to providethe desired effect, i.e., inhibit the formation of new blood vessels.The term “therapeutically effective amount” therefore refers to anamount of an anti-DEspR antibody or antigen-binding fragment thereofusing the methods as disclosed herein, that is sufficient to effect aparticular effect when administered to a typical subject. An effectiveamount as used herein would also include an amount sufficient to delaythe development of a symptom of the disease, alter the course of asymptom disease (for example but not limited to, slow the progression ofa symptom of the disease), or reverse a symptom of the disease. Thus, itis not possible to specify the exact “effective amount”. However, forany given case, an appropriate “effective amount” can be determined byone of ordinary skill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD₅₀/ED₅₀. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC₅₀ (i.e., theconcentration of the anti-DEspR antibody or antigen-binding fragmentthereof), which achieves a half-maximal inhibition of symptoms) asdetermined in cell culture, or in an appropriate animal model. Levels inplasma can be measured, for example, by high performance liquidchromatography. The effects of any particular dosage can be monitored bya suitable bioassay. The dosage can be determined by a physician andadjusted, as necessary, to suit observed effects of the treatment.

In other embodiments, the methods provided for inhibiting angiogenesisin a tissue of a subject or individual having a disease or disorderdependent or modulated by angiogenesis by administering to the subject atherapuetically effective amount of a composition comprising anangiogenesis-inhibiting amount of an anti-DEspR antibody orantigen-binding fragment thereof, can further comprise administrationone or more additional treatments such as angiogenic inhibitors,chemotherapy, radiation, surgery, or other treatments known to those ofskill in the art to inhibit angiogenesis.

In some embodiments, the methods described herein further compriseadministration of a combination of an anti-DEspR antibody orantigen-binding fragment thereof, with one or more additionalanti-cancer therapies. Examples of additional anti-cancer therapiesinclude, without limitation, surgery, radiation therapy (radiotherapy),biotherapy, immunotherapy, chemotherapy, or a combination of thesetherapies. In addition, cytotoxic agents, anti-angiogenic andanti-proliferative agents, targeted therapies like Erlotinib orimmunotherapy like OPDIVA®, can be used in combination with theanti-DEspR antibody or antigen-binding fragment thereof

In certain aspects of any of the methods and uses, the inventionprovides treating cancer by administering effective amounts of ananti-DEspR antibody or antigen-binding fragment thereof and one or morechemotherapeutic agents to a subject susceptible to, or diagnosed with,locally recurrent or previously untreated cancer. A variety ofchemotherapeutic agents can be used in the combined treatment methodsand uses of the invention. An exemplary and non-limiting list ofchemotherapeutic agents contemplated for use in the methods describedherein is provided under “Definition,” or described herein.

In some embodiments, the methods described herein compriseadministration of an anti-DEspR antibody or antigen-binding fragmentthereof with one or more chemotherapeutic agents (e.g., a cocktail) orany combination thereof. In some embodiments, the methods describedherein comprise administration of an anti-DEspR antibody orantigen-binding fragment thereof with a chemotherapeutic agent(s) as anantibody drug conjugate. Examples of chemotherapeutic agents includealkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN.™.);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines, such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics, such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin .gamma1and calicheamicin thetaI, see, e.g., Angew Chem. Intl. Ed. Engl. 33:183-186 (1994); dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antiobioticchromomophores), aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin;chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites, such as methotrexate and5-fluorouracil (5-FU); folic acid analogues, such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as, ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens, such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals, such asaminoglutethimide, mitotane, trilostane; folic acid replenisher, such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfomithine; elliptinium acetate; anepothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidamine; maytansinoids, such as maytansine and ansamitocins;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.®.;razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,e.g. paclitaxel (TAXOL.™., Bristol-Myers Squibb Oncology, Princeton,N.J.) and doxetaxel (TAXOTERE.®., Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors, such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); and anti-androgens,such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;siRNA and pharmaceutically acceptable salts, acids or derivatives of anyof the above. Other chemotherapeutic agents that can be used with thecompositions and methods described herein are disclosed in USPublication No. 20080171040 or US Publication No. 20080305044 and areincorporated in their entirety by reference. In certain embodiments, thechemotherapeutic agent is for example, capecitabine, taxane,anthracycline, paclitaxel, docetaxel, paclitaxel protein-bound particles(e.g., ABRAXANE™), doxorubicin, epirubicin, 5-fluorouracil,cyclophosphamide or combinations thereof therapy. As used herein,combined administration includes simultaneous administration, usingseparate formulations or a single pharmaceutical formulation, andconsecutive administration in either order, wherein preferably there isa time period while both (or all) active agents simultaneously exerttheir biological activities. Preparation and dosing schedules for suchchemotherapeutic agents can be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for chemotherapy are also described inChemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore,Md. (1992). Accordingly, in some embodiments, the chemotherapeutic agentcan precede, or follow administration of the anti-DEspR antibody orantigen-binding fragment thereof or can be given simultaneouslytherewith.

In some other embodiments of the methods described herein, othertherapeutic agents useful for combination tumor therapy with theanti-DEspR antibodies or antigen-binding fragments thereof describedherein include antagonists of other factors that are involved in tumorgrowth, such as EGFR, ErbB2 (also known as Her2), ErbB3, ErbB4, or TNF.In some embodiments, it can be beneficial to also administer one or morecytokines to the subject. In some embodiments, the anti-DEspR antibodyor antigen-binding fragment thereof is co-administered with a growthinhibitory agent. For example, the growth inhibitory agent can beadministered first, followed by the anti-DEspR antibody orantigen-binding fragment thereof. However, simultaneous administrationor administration of the anti-DEspR antibody or antigen-binding fragmentthereof first is also contemplated. Suitable dosages for the growthinhibitory agent are those presently used and can be lowered due to thecombined action (synergy) of the growth inhibitory agent and theanti-DEspR antibody or antigen-binding fragment thereof.

Examples of additional angiogenic inhibitors that can be used incombination with the DEspR inhibitors, such as anti-DEspR antibodies andantigen-binding fragments thereof, described herein include, but are notlimited to: direct angiogenesis inhibitors, Angiostatin, Bevacizumab(AVASTIN®), Arresten, Canstatin, Combretastatin, Endostatin, NM-3,Thrombospondin, Tumstatin, 2-methoxyestradiol, cetuximab (ERBITUX®),panitumumab (VECTIBIX™), trastuzumab (HERCEPTIN®) and Vitaxin; andindirect angiogenesis inhibitors: ZD1839 (Iressa), ZD6474, OS1774(Tarceva), CI1033, PKI1666, IMC225 (Erbitux), PTK787, SU6668, SU11248,Herceptin, and IFN-α, CELEBREX® (Celecoxib), THALOMID® (Thalidomide),and IFN-α.

In some embodiments, the additional angiogenesis inhibitors for use inthe methods described herein include but are not limited to smallmolecule tyrosine kinase inhibitors (TKIs) of multiple pro-angiogenicgrowth factor receptors. The three TKIs that are currently approved asanti-cancer therapies are erlotinib (TARCEVA®), sorafenib (NEXAVAR®),and sunitinib (SUTENT®).

In some embodiments, the angiogenesis inhibitors for use in the methodsdescribed herein include but are not limited to inhibitors of mTOR(mammalian target of rapamycin) such as temsirolimus (TORICEL™),bortezomib (VELCADE®), thalidomide (THALOMID®), and Doxycyclin,

In other embodiments, the angiogenesis inhibitors for use in the methodsdescribed herein include one or more drugs that target the VEGF pathway.Bevacizumab (AVASTIN®) was the first drug that targeted new bloodvessels to be approved for use against cancer. It is a monoclonalantibody that binds to VEGF, thereby blocking VEGF from reaching theVEGF receptor (VEGFR). Other drugs, such as sunitinib (SUTENT®) andsorafenib (NEXAVAR®), are small molecules that attach to the VEGFreceptor itself, preventing it from being turned on. Such drugs arecollectively termed VEGF inhibitors. As the VEGF/VPF protein interactswith the VEGFRs, inhibition of either the ligand VEGF, e.g. by reducingthe amount that is available to interact with the receptor; orinhibition of the receptor's intrinsic tyrosine kinase activity, blocksthe function of this pathway. This pathway controls endothelial cellgrowth, as well as permeability, and these functions are mediatedthrough the VEGFRs.

Accordingly, as described herein, “VEGF inhibitors” for use asangiogenesis inhibitors include any compound or agent that produces adirect or indirect effect on the signaling pathways that promote growth,proliferation and survival of a cell by inhibiting the function of theVEGF protein, including inhibiting the function of VEGF receptorproteins. These include any organic or inorganic molecule, including,but not limited to modified and unmodified nucleic acids such asantisense nucleic acids, RNAi agents such as siRNA or shRNA, peptides,peptidomimetics, receptors, ligands, and antibodies that inhibit theVEGF signaling pathway. The siRNAs are targeted at components of theVEGF pathways and can inhibit the VEGF pathway. Preferred VEGFinhibitors, include for example, AVASTIN® (bevacizumab), an anti-VEGFmonoclonal antibody of Genentech, Inc. of South San Francisco, Calif.,VEGF Trap (Regeneron/Aventis). Additional VEGF inhibitors includeCP-547,632 (3-(4-Bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin1-yl-butyl)-ureidol-isothiazole-4-carboxylic acid amide hydrochloride;Pfizer Inc. , NY), AG13736, AG28262 (Pfizer Inc.), SU5416, SU11248, &SU6668 (formerly Sugen Inc., now Pfizer, New York, N.Y.), ZD-6474(AstraZeneca), ZD4190 which inhibits VEGF-R2 and -R1 (AstraZeneca),CEP-7055 (Cephalon Inc., Frazer, Pa.), PKC 412 (Novartis), AEE788(Novartis), AZD-2171), NEXAVAR® (BAY 43-9006, sorafenib; BayerPharmaceuticals and Onyx Pharmaceuticals), vatalanib (also known asPTK-787, ZK-222584: Novartis & Schering: AG), MACUGEN® (pegaptaniboctasodium, NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862(glufanide disodium, Cytran Inc. of Kirkland, Wash., USA),VEGFR2-selective monoclonal antibody DC101 (ImClone Systems, Inc.),angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colo.) andChiron (Emeryville, Calif.), Sirna-027 (an siRNA-based VEGFR1 inhibitor,Sirna Therapeutics, San Francisco, Calif.) Caplostatin, solubleectodomains of the VEGF receptors, Neovastat (Æterna Zentaris Inc;Quebec City, Calif.), ZM323881 (CalBiochem. CA, USA), pegaptanib(Macugen) (Eyetech Pharmaceuticals), an anti-VEGF aptamer andcombinations thereof.

VEGF inhibitors are also disclosed in US Pat. 6,534,524 and 6,235,764,both of which are incorporated in their entirety. Additional VEGFinhibitors are described in, for example in WO 99/24440 (published May20, 1999), International Application PCT/IB99/00797 (filed May 3, 1999),in WO 95/21613 (published Aug. 17, 1995), WO 99/61422 (published Dec. 2,1999), U.S. Pat. Publ. No. 20060094032 “siRNA agents targeting VEGF”,U.S. Pat. No. 6,534,524 (discloses AG13736), U.S. Pat. No. 5,834,504(issued Nov. 10, 1998), WO 98/50356 (published Nov. 12, 1998), U.S. Pat.No. 5, 883,113 (issued Mar. 16, 1999), U.S. Pat. No. 5, 886,020 (issuedMar. 23, 1999), U.S. Pat. No. 5,792,783 (issued Aug. 11, 1998), U.S.Pat. No. 6,653,308 (issued Nov. 25, 2003), WO 99/10349 (published Mar.4, 1999), WO 97/32856 (published Sep. 12, 1997), WO 97/22596 (publishedJun. 26, 1997), WO 98/54093 (published Dec. 3, 1998), WO 98/02438(published Jan. 22, 1998), WO 99/16755 (published Apr. 8, 1999), and WO98/02437 (published Jan. 22, 1998), WO 01/02369 (published Jan. 11,2001); U.S. Provisional Application No. 60/491,771 piled Jul. 31, 2003);U.S. Provisional Application No. 60/460,695 (filed Apr. 3, 2003); and WO03/106462A1 (published Dec. 24, 2003). Other examples of VEGF inhibitorsare disclosed in International Patent Publications WO 99/62890 publishedDec. 9, 1999, WO 01/95353 published Dec. 13, 2001 and WO 02/44158published Jun. 6, 2002.

In other embodiments, the angiogenesis inhibitors for use in the methodsdescribed herein include anti-angiogenic factors such as alpha-2antiplasmin (fragment), angiostatin (plasminogen fragment),antiangiogenic antithrombin III, cartilage-derived inhibitor (CDI), CD59complement fragment, endostatin (collagen XVIII fragment), fibronectinfragment, gro-beta (a C-X-C chemokine), heparinases heparinhexasaccharide fragment, human chorionic gonadotropin (hCG), interferonalpha/beta/gamma, interferon inducible protein (IP-10), interleukin-12,kringle 5 (plasminogen fragment), beta-thromboglobulin, EGF (fragment),VEGF inhibitor, endostatin, fibronection (45 kD fragment), highmolecular weight kininogen (domain 5), NK1, NK2, NK3 fragments of HGF,PF-4, serpin proteinase inhibitor 8, TGF-beta-1, thrombospondin-1,prosaposin, p53, angioarrestin, metalloproteinase inhibitors (TIMPs),2-Methoxyestradiol, placental ribonuclease inhibitor, plasminogenactivator inhibitor, prolactin 16kD fragment, proliferin-related protein(PRP), retinoids, tetrahydrocortisol-S transforming growth factor-beta(TGF-b), vasculostatin, and vasostatin (calreticulinfragment).pamidronate thalidomide, TNP470, the bisphosphonate familysuch as amino-bisphosphonate zoledronic acid. bombesin/gastrin-releasingpeptide (GRP) antagonists such as RC-3095 and RC-3940-II (Bajol A M, et.al., British Journal of Cancer (2004) 90, 245-252), anti-VEGF peptideRRKRRR (dRK6) (Seung-Ah Yoo, J.Immuno, 2005, 174: 5846-5855).

Thus, in connection with the administration of anti-DEspR antibodies andantigen-binding fragments thereof, a compound which inhibitsangiogenesis indicates that administration in a clinically appropriatemanner results in a beneficial effect for at least a statisticallysignificant fraction of patients, such as improvement of symptoms, acure, a reduction in disease load, reduction in tumor mass or cellnumbers, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating the particular type of disease or condition.

Examples of additional DEspR inhibitors include, but are not limited to,molecules which block the binding of VEGFsp (sp26, sp17), ET-1 and/orother ET-1 orVEGFsp-like ligands to DEspR, compounds which interferewith downstream signaling events of DEspR, or other compounds or agentsthat inhibit activation of the receptor. Such compounds can bind toDEspR and prevent binding of VEGFsp (sp26, sp17), ET-1 or other mimeticligands. Other inhibitors including small molecules that bind to theDEspR domain that binds to VEGFsp, soluble DEspR receptors, peptidescontaining the DEspR ET-1 and/or VEGFsp binding domains, etc. are alsocontemplated. For example, in some aspects, provided herein areVEGFsp-26 peptides with one or more modifications that stabilize thepeptide in vivo to be used in methods of inihibiting DEspR expressionand/or function.

The compositions described herein can also contain more than one activecompound as necessary for the particular indication being treated, andthese active compounds are preferably those with complementaryactivities that do not adversely affect each other. For example, it canbe desirable to further provide antibodies or antagonists that bind toEGFR, VEGF, VEGFR, or ErbB2 (e.g., Herceptin™). Alternatively, or inaddition, the composition can comprise a cytotoxic agent, cytokine,growth inhibitory agent and/or VEGFR antagonist. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended.

In certain aspects of any of the methods and uses described herein,other therapeutic agents useful for combination cancer therapy with theantibody of the invention include other anti-angiogenic agents. Manyanti-angiogenic agents have been identified and are known in the arts,including those listed by Carmeliet and Jain (2000). In someembodiments, the DEspR antagonist, such as a humanized anti-DEspRantibody or antigen-binding fragment thereof described herein is used incombination with a VEGF antagonist or a VEGF receptor antagonist such asVEGF variants, soluble VEGF receptor fragments, aptamers capable ofblocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, low moleculeweight inhibitors of VEGFR tyrosine kinases and any combinationsthereof. Alternatively, or in addition, two or more anti-DEspRantagonists can be co-administered to the subject.

For the treatment of diseases, as described herein, the appropriatedosage of an anti-DEspR antibody or antigen-binding fragment thereofwill depend on the type of disease to be treated, as defined above, theseverity and course of the disease, whether the anti-DEspR antibody orantigen-binding fragment thereof is administered for preventive ortherapeutic purposes, previous therapeutic indications, the subject'sclinical history and response to the anti-DEspR antibody orantigen-binding fragment thereof, and the discretion of the attendingphysician. The anti-DEspR antibody or antigen-binding fragment thereofis suitably administered to the subject at one time or over a series oftreatments. In a combination therapy regimen, the anti-DEspR antibody orantigen-binding fragment thereof and the one or more anti-cancertherapeutic agents described herein are administered in atherapeutically effective or synergistic amount. As used herein, atherapeutically effective amount is such that co-administration of ananti-DEspR antibody or antigen-binding fragment thereof and one or moreother therapeutic agents, or administration of a composition describedherein, results in reduction or inhibition of the cancer as describedherein. A therapeutically synergistic amount is that amount of ananti-DEspR antibody or antigen-binding fragment thereof and one or moreother therapeutic agents necessary to synergistically or significantlyreduce or eliminate conditions or symptoms associated with a particulardisease. In some cases, the anti-DEspR antibody or antigen-bindingfragment thereof can be co-administered with one or more additionaltherapeutically effective agents to give an additive effect resulting ina significantly reduction or eliminatation of conditions or symptomsassociated with a particular disease, but with a much reduced toxicityprofile due to lower dosages of one or more of the additionaltherapeutically effective agents.

The anti-DEspR antibody or antigen-binding fragment thereof and the oneor more other therapeutic agents can be administered simultaneously orsequentially in an amount and for a time sufficient to reduce oreliminate the occurrence or recurrence of a tumor, a dormant tumor, or amicrometastases. The anti-DEspR antibody or antigen-binding fragmentthereof and the one or more other therapeutic agents can be administeredas maintenance therapy to prevent or reduce the likelihood of recurrenceof the tumor.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents or other anti-cancer agentswill be generally around those already employed in clinical therapies,e.g., where the chemotherapeutics are administered alone or incombination with other chemotherapeutics or targeted therapies.Variation in dosage will likely occur depending on the condition beingtreated. The physician administering treatment will be able to determinethe appropriate dose for the individual subject.

In addition to the above therapeutic regimes, the subject can besubjected to radiation therapy.

In certain embodiments of any of the methods, uses and compositionsdescribed herein, the administered DEspR antibody is an intact, nakedantibody. However, in some embodiments, the anti-DEspR antibody can beconjugated with a cytotoxic agent. In certain embodiments of any of themethods and uses, the conjugated anti-DEspR antibody and/or DEspRantigen-binding fragment thereof is/are internalized by the cell,resulting in increased therapeutic efficacy of the conjugate in killingthe cancer cell to which it binds. In some embodiments, the cytotoxicagent conjugated to the DEspR antibody and/or DEspR antigen-bindingfragment thereof targets or interferes with nucleic acid in the cancercell. Examples of such cytotoxic agents include maytansinoids,calicheamicins, ribonucleases and DNA endonucleases, and are furtherdescribed elsewhere herein.

This invention is further illustrated by the following examples whichshould not be construed as limiting. It is understood that the foregoingdescription and the following examples are illustrative only and are notto be taken as limitations upon the scope of the invention. Variouschanges and modifications to the disclosed embodiments, which will beapparent to those of skill in the art, may be made without departingfrom the spirit and scope of the present invention. Further, allpatents, patent applications, and publications identified are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents are based on the information availableto the applicants and do not constitute any admission as to thecorrectness of the dates or contents of these documents.

EXAMPLES Example 1

Peptide GSNEMKSRWNWGS (SEQ ID NO: 1) was used as antigenic peptide togenerate monoclonal antibodies directed to DEspR. A smaller or partialpeptide EMKSRWNWGS (SEQ ID NO: 2) was then used to screen (by ELISA) formonoclonal antibodies and narrow down the potential epitope for 6G8G7.

The stroke prone Tg25+ rat model was developed in the polygenic DahlSalt-sensitive hypertensive strain, transgenic for human cholesterylester transfer protein conveying a hyperlipidemic profile (Herrera etal. 1999). These rats were made stroke prone by early life Na-exposureduring gestation to PURINA 5001 regular rat chow with 0.3% NaCl (Decanoet al. 2009). Anatomical and histological analysis revealed that Tg25+stroke prone rats exhibited parenchymal hemorrhages (FIGS. 1A-1B) andhemorrhagic infarctions (FIG. 1C).

The rat stroke model exhibits stroke-pathology lesions seen in humans.These lesions were detectable on ex vivo 11.7T MR-imaging using gradientecho sequences (FIG. 2A) and T2weighted MRI (FIGS. 2B-2C). Ischemiasurrounding microhemorrhages were noted on T2-weighted intensityanalysis (FIG. 2C) and on analysis of T2 relaxation time (FIGS. 2D-2E).

As observed in humans, stroke-prone Tg25+ females exhibited earlieronset of ischemic-hemorrhagic strokes compared to male Tg25+ rats (FIG.3) (Decano et al. 2009).

At onset of acute stroke, rats exhibit neurologic deficits such asseizures, paresis or paralysis, or dystonic movements, or lethargy. Whenthese neurologic deficits appear, rats are assigned to either theanti-DEspR (anti-rat DESpR monoclonal antibody, 10a3h10) treatment groupor the control isotype antibody group. One dose of 30 μg/kg isadministered intravenously. Rats were then monitored and aided to haveadequate food and water until they recover or needed to be euthanized Asshown in FIG. 4, early anti-DEspR mAb therapy at acute stroke resolvespresenting neurologic deficits and increases survival of spTg25+ femalerats.

Without wishing to be bound or limited by theory, a putative mechanismfor the results described herein includes stabilizing leaky microvesselsor microvascular disruption in stroke ischemic sites, as seen in cancer.

Treatment of rats with spontaneous breast tumors with anti-DEspR therapy(10a3h10 antibody) resulted not just in decreased tumor growth, but alsostabilization of ‘tumor leaky neovessels.’ As seen in FIG. 6A,pretreatment, rat tumor vessels are eroded, with red blood cellsencroaching into the tumor, and tumor cells encroaching into the vessellumen. After a 4-dose therapy over several weeks, tumors from treatedrats exhibited smaller sizes as well as tumor blood vessels with intactendothelium (FIG. 6B).

As demonstrated herein, DEspR is a membrane bound receptor that isglycosylated, and pulled down' with galectin-1 as identified by MassSpectronomy peptide signature analysis. Galectin-1 has recently beenimplicated to ‘tie-up” the key receptor for VEGF, VEGF-R2, in themembrane as a mechanism for VEGF-resistance, (Croci et al 2014. Cell156(4):744-58), however, no pulldown experiments were reported by Crociet al 2014. Anti-human DEspR mAb, 5g12e8 monoclonal antibody, was usedfor pulldown of DEspR from membrane proteins isolated from glioblastomau87 CSCs and from Cos1-DEspR permanent cell transfectants.

As shown in FIG. 7, hDEspR Cos-1 cell permanent transfectants expressthe DEspR protein produced by a recombinant DEspR-unspliced minigeneconstruct, which automated sequencing reports to contain the purported‘stop codon sequence of TGA’ at amino acid #14 position. The minigeneconstruct contains the unspliced cDNA construct. As demonstrated herein,detection of protein products >10 kDa by anti-DEspR monoclonal antibody5g12e8 indicates unequivocally that the stop codon is not present. Astop codon would produce only a 13-aa long peptide which is only 1.547kDa. Detection of identical pulldown DEspR products (band detected byanti-DEspR mAb) in human glioblastoma U87 cells and DEspR+ Cos 1-celltransfectants indicates that the unspliced hDEspRminigene construct istranscribed, spliced, translated and undergoes post-translationalglycosylation. There is less galectin-1 in Cos1 cells than U87 cells;non-transfected Cos1 cells have no detectable DEspR by western blot andbinding assays. The consistent pulldown of DEspR-Galectin1 complex (n>5)indicates that DEspR could underlie the persistent angiogenesis observedin so called “VEGF-resistance” with galectin-1 serving as the scaffoldand linker for different glycosylated proteins.

5G12E8 and 6G8G7 monoclonal antibodies work in western blots and wereused to confirm DEspR protein in pulldown (FIGS. 8A and 8B: ˜17 kDaprotein band is DEspR-glycosylated (as shown by PNGase digest, and ˜12.5kDa protein is DEspR with less glycosylation. Two different monoclonalantibodies raised against different domains in the DEspR protein bind tothe identical protein bands on Western blot of pull-down proteins (FIGS.8A, 8B), thus refuting the existence of a stop codon at amino acid #14position and demonstrating DEspR protein expression in human cancercells.

Western blot ‘walking’ with the 5G12E8 monoclonal antibody binding toamino terminal end, and 6G8G7 binding to the predicted ligand-bindingdomain demonstrate the existence of DEspR. If the stop codon were indeedpresent at position amino acid 14 W (□), then 6G8G7 should not bind tothe identical 17 kDa and 12.5 kDa bands, or any protein. In addition,7C5B2 monoclonal antibody does not ‘work’ in Western Blot analysisindicating that conformation plays a role in its epitope in the 9-aaN-terminal peptide antigen. It is to be noted that the peptide used togenerate 6G8G7 is 100% identical in Human-Rat-Mouse (unlike peptide for7C5B2 and 5G12E8), thus facilitating FDA-required 2-species toxicitystudies.

DEspR, a single transmembrane integral membrane protein, was notdetectable by peptide mass fingerprinting methods using techniques suchas MALDI-TOF Mass Spectrometry analysis (do D. Pappin, Cold SpringHarbor, c/o CRO). This is not surprising given that majority (87-97%) ofintegral membrane proteins are not identifiable on Mass Spectrometry,and only >150kDa integral membrane proteins (with e2 transmembranedomains) were detected by MS.

Bensalem et al. 2006: Peptide mass fingerprinting of membrane proteins,using techniques such as MALDI-TOF MS, remains a ‘real challenge for atleast 3 reasons: 1. Membrane proteins are naturally present at lowlevels. 2. Most of the detergents strongly inhibit proteases and havedeleterious effects on MALDI spectra. 3. Despite the presence ofdetergent, membrane proteins are unstable and often aggregate [BensalemN, et al., High sensitivity identification of membrane proteins by MALDITOF-MASS Spectrometry using polystyrene beads. J Proteome Res 2007,6:1595-1602.] Mirza et al 2007 at the National Center for ProteomicsResearch, Medical College of Wisconsin, detected only 204 (3% of 6718)integral membrane proteins from rat endothelial cells even after theircholoroform-extraction method. Notably, no VEGFR2 was identified onMS-like DEspR. [Mirza S P Halligan B D, Greene A S, Olivier M. 2007.Improved method for the analysis of membrane proteins by massspectrometry. Physiol Genomics 30:89-94, 2007. Peng et al 2011 detectedonly 301 (4.5% of 6718) integral membrane proteins via SDS-PAGE shotgunproteomics. [Peng L, Kapp E A, McLauhlan D Jordan T W. Characterizatinof the Asia Oeania human proteome organization membrane proteomicsinitiative standard using SDS-PAGE shotgun proteomics. Proteomics11:4376-4384.]. Fagerberd et al 2010: range of integral membraneproteins 5508 to 7651 depending on the method used. Based on a majoritydecision method, estimate is 5539 human genes code for membrane proteinsor 26% of human genome. Highest count using SCX-RPLC-MS/MS (MudPIT)strategy detected 876 integral membrane proteins or 13% of 6718 integralmembrane proteins in murine NK cells [Fagerberd L, Jonasson K, vonHeijneG, Uhlen M, Berglund L. 2010. Prediction of the human membrane proteome.Proteomics 10:1141-1149; Blonder J et al., J Proteome Res 2004, 3,862-870.] Almen et al 2009, mined the human proteome and identified themembrane proteome subset using 3 prediction tools for alpha-helices:Phobius, TMHMM, and SOSUI. This data set was reduced to a non-redundantset by aligning it to the human genome and then clustered using theISODATA algorithm. 6,718 human membrane proteins were identified (32% ofall human proteins—estimated at 21,000 proteins)—901 are GPCRs and7-transmembrane domain receptors, 88% of the rest aresingle-transmembrane domain receptors. [Almen 2009, Fagerbered et al2010.] DEspR has single transmembrane domain. This is concordant withthe most recent and reliable set of genes in the human genome whichlists 5,359 validated protein coding α-helical transmembrane proteins˜27% of the entire human proteome.

Single transmembrane proteins—like DEspR and VEGF-R2—are not detected onMass Spectrometry analysis of membrane proteins. Zhou et al 2011.Moreover, of the ones detected by Mass Spectrometry using an improvedmethod that detects more membrane proteins using a centrifugal proteomicreactor, all membrane proteins detected by Mass Spectrometry were >150kDa and contained e 2 transmembrane domains. [Zhou H, Wang F, Wang Y,Ning Z, Hou W, Wright T G, Sundaram M, Zong S, Yao Z, Figeys D. 2011.Improved recovery and identification of membrane proteins from rathepatic cells using a centrifugal proteomic reactor. Mol Cell.Proteomics 10.10 (2011). Cao et al, 2013: “Deglycosylation of plasmamembrane proteins by treatment with PNGase-F did not yield detection ofadditional hydrophobic proteins” (by MS with in-gel proteolyticpredigestion) (Cao L, Clifton J G, Reutter W, Josic D. 2013. Massspectrometry-based analysis of rat liver and hepatocellular carcinomaMorris hepatoma 7777 plasma membrane proteome. Analytical Chem85:8112-8120, 2013.)Therefore, peptide mass fingerprinting massspectrometry analysis of PNGase-treated pulldown proteins, which did notidentify DespR, does not negate the existence of DEspR protein.

Based on UNIPROT criteria for determining a protein's existence, thedemonstration of protein-protein interactions (as in the DEspR-galectin1 complex) and the antibody detection of DEspR protein in human tumorcells and endothelial cells in vitro using multiple techniques(immunofluorescence analysis of cells and tumor tissue arrays—breastcancer, pancreatic cancer, glioblastoma, stomach, colon, ovariancancers)—each fulfill established criteria for documenting the existenceof DEspR protein in human cells. Together, these date provide multiplelines of evidence for DEspR protein at the highest criterion-level ofprotein determination.

Immunostaining of human tissue using 7C5B2 monoclonal antibody has beendone. Furthermore, based on UNIPROT criteria, functional consequences ofinhibition of DEspR by anti-DEspR antibody indicate the existence of afunctional protein. Demonstration of DEspR protein is concordant withdemonstration of spliced and unspliced DEspR-mRNA by ARMS (Herrera et al2014). DEspR (17K glycosylated) and Galectin-1 (14.5 kDa) are distinctproteins. The consistent pulldown of galectin1 does not suggest that theanti-DEspR monoclonal antibody detects Galectin1 and that there is noDEspR protein. No cross reactivity of antibodies has been detected.While both DEspR and galectin-1 are found at the plasma membrane, in thecytosol and nucleus concordant with colocalization for complexing,galectin1 is secreted from the cell into the ECM, but not DEspR. DEspRis 17 kDa (glycosylated) and 12.5 kDa (non or minimal glycosylation).PNGase digest shows decreasing MW of DEspR 17 kDa band (FIG. 9). WhileDEspR is glycosylated, galectin1 is not glycosylated. “Galectin-1 is nota glycosylated protein” (Cherch et al 2006. Glycobiolgy16:137R-157R)—hence the 17 kDa glycosylated protein detected on WBanalysis proven by PNG-ase digestion cannot be galectin1. Anti-DEspR5G12E8 monoclonal antibody does not bind galectin-1 on western blotanalysis of pulldown proteins, and does not bind recombinant galectin-1protein on western blot (FIGS. 10A-10B).

Concordant with pulldown of a DEspR-galectin1 complex, DEspR andgalectin1 colocalize in tumor cells at the invasive front of a humanglioblastoma (U87-csc) xenograft subcutaneous tumor. Detection ofcolocalization confirms pulldown data identifying a DEspR-galectin1complex. Since Croci et al. identified galectin-1 as a mechanism foranti-VEGF (Avastin) resistance, the DEspR-galectin-1 complex defines amechanism for the observed resistance through DEspR-mediatedangiogenesis. Since galectin-1 is increased in cancers, and has beenimplicated in metastasis and tumor angiogenesis, inhibition of DEspRcould then impact the DEspR-galectin-1 complex, thus expanding themechanisms for efficacy from solo-DEspR-mediated to also includeDEspR-galectin1 complex-mediated mechanisms. Hsu et al. 2013. Galectin-1promotes lung cancer tumor metastasis by potentiating integrin α6β4 andNotchl/Jagged2 signaling pathway. Carcinogenesis Feb. 6, 2013.

Protein-glycan interactions play important functions in several aspectsof cancer biology, including cancer transformation, growth, metastasis,angiogenesis and immune response. Galectin-1, the first proteindiscovered in the family, has been shown to be overexpressed in manymalignancies, including lymphoma, oral, colon, bladder, ovarian,astrocytoma, liver, pancreatic and melanoma carcinomas. Dysregulation ofgalectin-1 in cancer has also been correlated with the aggressiveness ofthese tumors. Rek,A. et al. (2009) Therapeutically targetingprotein-glycan interactions. Br. J. Pharmacol., 157, 686-694;Yamamoto-Sugitani, M. et al. (2011) Galectin-3 (Gal-3) induced byleukemia microenvironment promotes drug resistance and bone marrowlodgment in chronic myelogenous leukemia. Proc. Natl. Acad. Sci. U.S.A.,108, 17468-17473; Wu,H. et al. (2012) Overexpression of galectin-1 isassociated with poor prognosis in human hepatocellular carcinomafollowing resection. J. Gastroenterol. Hepatol., 27, 1312-1319; Xue,X.et al. (2011) Galectin-1 secreted by activated stellate cells inpancreatic ductal adenocarcinoma stroma promotes proliferation andinvasion of pancreatic cancer cells: an in vitro study on themicroenvironment of pancreatic ductal adenocarcinoma. Pancreas, 40,832-839; Watanabe, M. et al. (2011) Clinical significance of circulatinggalectins as colorectal cancer markers. Oncol. Rep., 25, 1217-1226;Kamper, P. et al. (2011) Proteomic analysis identifies galectin-1 as apredictive biomarker for relapsed/refractory disease in classicalHodgkin lymphoma. Blood, 117, 6638-6649.

A lead mouse prototype anti-human DEspR monoclonal antibody (7C5B2 or7c5b2) has been characterized to inhibit pancreatic ductaladenocarcinoma (PDAC) and glioblastoma U87 cancer stem cells in vitroand in vivo in CSC-dervied xenograft tumors in nude rats.DEspR-inhibition in vivo and in vitro inhibit CSC anoikis resistance,tumor growth, tumor vasculo-angiogenesis (Herrera et al 2014, PloSOne).A lead mouse prototype anti-humanDEspR monoclonal antibody (7c5b2) is“competed out” by the known ligands for human DEspR: endothelin-1 (ET1)and VEGFsp17, and VEGFsp26. (FIG. 13). This shows specificity for7c5b2-antibody binding to human DEspR such that the antibody blocks thebinding of DEspR-ligands, ET1 and VEGFsp. Two peptides of differinglengths: VEGFsp26 with anti-angiogenic function, and VEGFsp17 withpro-angiogenic function.

These observations indicate that 7c5b2 binds DEspR and blocks ligandengagement of DEspR for both its ligands—ET1 and VEGFsp (VEGFsp17-aa andVEGFsp26-aa). 7c5b2 has been sequenced and a fully human compositeantibody developed (Antitope, UK; Lake Pharma, USA). ELISA and growthcapabilities selected several candidates: vh5/vk1, vh3/vk2, andhumanized forms of HV2KV2 (SEQ ID NOs: 20 and 50).

Mouse monoclonal anti-hDEspR antibodies, 7C5B2, 5G12E8 (N-terminalpeptide antigen), and 6G8G7 (binding domain peptide antigen) havecomparable in vitro inhibition of CSC growth in suspension cultures(FIG. 14). Growth in suspension cultures requires anoikis resistance,and is a stem cell characteristic. Anti-hDEspR inhibition results indecreased CSC growth in different human tumor cell lines: colon cancer(HCT116), pancreatic ductal adenocarcinoma (Panc1), lung cancer (H460),triple negative breast cancer (MB231) and glioblastoma (U87).

Efficacy in different tumor cell line-derived CSCs indicates that DEspRplays a key role in CSC growth in suspension cultures, ie,unattached—which then indicates key roles in metastasis as metastaticcancer cells need to survive detachment or be anoikis resistant.

Comparative analysis of mouse prototype 7C5B2 (amino terminal endantibody) and 6G8G7 (binding domain antibody) have comparable Kd 5.2ug/ml or 33 nM for binding to DEspR+ pancreatic cancer Panc1 cells.Fully human VH5/VK1 has a slightly better Kd than VH3/VK2 fully humancomposite antibodies. When adjusted for Kd, equivalent dosing ofanti-DEspR antibodies shows equivalent efficacy of VH5/VK1 [fully humancomposite antibody] to mouse prototype monoclonal variant antibodies7C5B2 and 6G8G7. In vitro assays show that VH5/VK1 inhibits glioblastomaCSC survival and growth in detached suspension culture conditions betterthan VH3/VK1. Notably, VH3/VK1 also exhibits inhibitory functionality(P<0.001). (FIG. 16).

In vivo analyses of inhibition of tumour growth in a heterotopicxenograft glioblastoma tumour model indicate that mouse prototype lead7C5B2 and its corresponding fully human anti-hDEspR monoclonal antibodyVH5/VK1 show that both inhibit tumour growth of different xenograftheterotopic (subQ) tumour models significantly developed from pancreaticcancer Panc1-CSCs, and triple negative breast cancer MB231-CSCs andMB468-CSCs (FIG. 17). Notably, VH5/VK1 performs equivalently to 7C5B2mouse prototype anti-DEspR monoclonal antibody. Data support VH5/VK1 asa therapeutic lead for anti-DEspR therapy. In vivo analyses ofanti-hDEspR-mediated inhibition of tumour growth in a heterotopicxenograft glioblastoma tumour model indicate that mouse prototype 5G12E8(used for pulldowns) and fully human anti-hDEspR lead VH5/VK1 inhibittumour growth from glioblastoma U87 CSCs significantly. (FIG. 18). SubQxenograft tumour models allow analysis of tumour progression to largertumour sizes and for longer duration compared to the orthotopicintracranial xenograft glioblastoma tumour models wherein nude rats withbrain tumors need to be euthanized within 30 days or less due toneurologic deficits and tumour masses of only 1000 mm3. Notably, VH5/VK1performs better than 5G12E8 monoclonal antibody (used for pulldown) ininhibiting tumour growth in the xenograft human glioblastoma U87heterotopic tumour model. Thus, the data described herein supportVH5/VK1 as a therapeutic lead for anti-DEspR therapy

Significant inhibition of U87 CSC-derived subcutaneous tumourprogression by 5G12E8 and VH5/VK1 monoclonal antibodies was observed. *P<0.05; *** P<0.001 (Two Way ANOVA followed by Student-Newman-Keuls Testfor multiple comparisons). In vivo analyses of inhibition of tumourinitiation in xenograft peritoneal metastatic models of pancreaticcancer (Panc1-CSCs) and of colon cancer (pilot data on HCT-116) indicatethat mouse prototype 5G12E8 (used for pulldowns) and fully humananti-hDEspR lead VH5/VK1 monoclonal antibody inhibit tumour initiationof peritoneal metastasis seeding and progression in at least 50% ofanimals seeded with 2×10⁶ CSCs. (FIGS. 19A-19C).

Data indicate clinical application of adjuvant therapy to preventperitoneal metastasis for PDAC and colon cancer. Data demonstrateinhibition of tumor initiation in vivo by VH5/VK1-Rx, experimentalmodelling of clinical application as adjuvant anti-DEspR therapy. Weobserved a significant increase in survival of nude rats injected with5G12E8-treated Panc1 CSCs (A, P=0.042); VH5/VK1-treated Panc1 CSCs (B,P=0.041) and VH5/VK1-treated HCT116 CSCs (C, P=0.08). P values fromKaplan-Meier Survival Analysis, Log-Rank Test. Note: 4/8 5G12E8-treatedrats were still alive after 120 days post-cell injection in experiment A(no tumours at euthanasia); 2/5 VH5/VK1-treated rats were still aliveafter 87 days post-cell injection in experiment B (no tumours ateuthanasia); half VH5/VK1-treated rats were still alive after 87 dayspost-cell injection in experiment C, and were electively euthanizedPost-mortem analysis revealed few tumours at euthanasia (FIGS. 19A-19C).

Additional data on survival analysis further indicate clinicalapplication to inhibit NSCLC (non-small cell lung cancer) tumorprogression (FIGS. 20A-20B, anti-DEspR treated subjects vs controls,P=0.002) and pancreatic peritoneal metastatic tumor progression (FIGS.21A-21B, anti-DEspR subjects vs Controls, P<0.01). Of note, anti-DEspRinhibition was significantly more efficacious than Gemcitabine treatment(Gemcitabine vs Controls, P=not significant), the gold standard fortreatment of pancreatic cancer.

Example 2

As described herein, variable heavy (V_(H)) and variable light (V_(L))chains sequences were obtained from the hybridoma 6g8g7 both at the DNAlevel by PCR and by Mass Spectrometry sequencing from 2 independentCROs. It was found that there were two variable heavy (V_(H)) and threevariable light (V_(L)) chains sequences.

Humanized chimeric sequences were made of the 6g8g7 murine monoclonalantibody CDR sequences with human IgG1/kappa Fc region.

Several 6g8g7-derived human monoclonal antibody candidates were nextdesigned with the following specifications:

a) CDR sequences from 6G8G7 HV2 (SEQ ID NOs: 14, 15, and 16) and 6G8G7KV1 (SEQ ID NOs: 28, 29, and 30)that retain 6G8G7 binding to rat andhuman DEspR.

b) Sequence modifications in variable regions were made to exclude orreduce the following sites for optimal biotherapeutic properties forefficacy, safety and manufacture: 1) potential CD4+ T-cell epitopes forlow immunogenicity; 2) acid-labile sites for antibody stability duringprocessing requiring low pH; 3) destabilizing post-translationalmodification sites predisposed to deamidation and oxidation forstructural stability of humabs and to retain functionality in adversemetabolic conditions such as in hypoxic tumors; and 4)post-translational modification sites predisposed to inappropriateN-glycosylation, isomerization, and pyroglutamate formation forreproducible production and efficacious performance.

c) Human IgG4 constant region was used to avail of IgG4 Fc's property ofinsignificant effector functions (i.e., minimal antibody-dependent cellcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)), butwith the S228P hinge-stabilizing mutation in order to minimize Fab armexchange (FAE) in vivo which would result in loss of therapeuticefficacy (Silva et al. 2015). The S228P mutation confers stabilityproperties to human IgG4. It is further noted that this S228Phinge-stabilized IgG4 is used in FDA-approved PD1 receptor inhibitorKeytruda, consistent with the S228P IgG4 as being suitable forbiotherapeutic applications. (Yang X et al. 2015).

Multiple 6G8G7-V_(H) (n=2) and 6G8G7-V_(L) (n=3) recombinant clones wereconstructed, sequenced to confirm the different constructs, andtransiently expressed in HEK293. Humanized antibodies were tested byELISA for binding to the antigenic peptide (representing thehuman/monkey/rat DEspR epitope). Some did not show significant binding,but others showed binding to the 6G8G7 antigenic peptide. The top twowere selected and then grown in 0.3 L medium to generate sufficientamounts for further characterization.

Next, the EC50 of binding to the antigenic peptide was determined byELISA, as shown in FIGS. 35A-35B, comparing two IgG4 6g8g7 humanizedantibody candidates (including humab1 comprising SEQ ID NOs: 63 and 65and humab2 comprising SEQ ID NO: 65 with substitutions in SEQ ID NO: 26at Kabat residues 83V and 85Y to give 83D and 85Y), and the IgG16G8G7humanized monoclonal antibody (comprising SEQ ID NOs: 61 and 65),and 6G8G7 murine monoclonal antibody (“6g8 mumab”).

These studies showed the following EC50 and Bmax for binding to theantigenic peptide detected on ELISA (Table 1). As shown in Table 1,based on Bmax, a 6g8g7-derived IgG4 humanized monoclonal antibody 1(“6g8^(IgG4) humab1”) is better than a 6g8g7-derived IgG4 humanizedmonoclonal antibody 2 (“6g8^(IgG4) humab2”) in binding to the antigenicpeptide on ELISA. Hence 6g8^(IgG4) humab1 was expressed in 0.5 L mediumfor further study and demonstration of functional activity of 6g8^(IgG4)humab as a lead candidate. This 0.5 L transient expression oftransfectants produced 110 mg of 6g8^(IgG4) humab.

TABLE 1 6g8^(IgG4) 6g8^(IgG1) 6g8^(IgG4) 6g8- Table 1. humab1 humab1humab2 mumab ELISA (AP) EC₅₀ (ug/ml) 11.7 ± 2.2  13.4 ± 2.5 4.8 ± 1.22.96 ± 0.93 Bmax (A₄₅₀) 2.62 ± 0.16 3.09 ± 0.2 1.31 ± 0.08 6.36 ± 0.48

Next, the EC50 binding to DEspR on intact cells at 4° C. was determinedto eliminate non-specific endocytosis. Binding was determined by FACSanalysis. As shown in FIG. 36, the 6g8^(IgIG4) humab1 exhibited betterbinding affinity to intact DEspR+ human cells than the original 6g8g7murine monoclonal antibody.

These studies show the following EC50 for binding to intact DEspR+cells. (Table 2) As shown in Table 2, the 6g8^(IgG4) humabl shows betterEC50 binding to intact cells than 6g8g7 murine monoclonal antibody fromwhich it is derived.

TABLE 2 Table-2. Binding to DEspR on intact human cells (FACS)6g8^(IgG4) 6g8^(IgG1) 6g8^(IgG4) 6g8 humab1 humab humab2 mumab EC50 0.64 ± 0.25  1.3 ± 0.74 ND 5.39 ± 1.84 (ug/ml) EC50 (nM) 21.1 ± 8.342.9 ± 24  ND 177.9 ± 60   Bmax (% + 31.5 ± 2.6 21.6 ± 3.0 ND 35.6 ±4.0  cells)

To confirm binding, we obtained a FACS fluorescence intensity plot of6g8^(IgG4) humab1 binding to DEspR on intact human cells (FIG. 37). The6g8^(IgG4) humab1 lead candidate was labeled with a ‘red’ fluorophore,AF568, and used for FACS analysis of DEspR+ human cells compared tocontrol IgG4 isotype.

Functionality of the lead candidate humab, 6g8^(IgG4) humab1 comprisingSEQ ID NOs: 63 and 65, was next determined. It was first tested whetheranti-DEspR 6g8^(IgG4) humab1 can inhibit neutrophil survival pertinentto stopping neutrophil-mediated blood brain barrier disruption thatcontributes to hemorrhagic conversion in stroke. Rat neutrophils wereobtained from stroke-prone rats. As shown in FIG. 38, the lead candidatehumab, 6g8^(IgG4) exhibited greater efficacy in vitro in inhibitingneutrophil survival compared to the original 6g8 mumab control.

The EC50 values shown in Table 3 demonstrate superior functionalactivity of the lead candidate 6g8^(IgG4)humab1 in the inhibition ofneutrophil survival compared to the 6g8 mumab. Notably, 6g8^(IgG4)humab1 (serving as reference) also exhibited better functional activityin inhibiting neutrophil survival than the 6g8 mumab.

TABLE 3 Table 3. Rat Neutrophil survival 6g8^(IgG4) 6g8^(IgG4) humab16g8^(IgG1) humab humab2 6g8 mumab IC₅₀ (ug/ml) 1.16 ± 0.3  6.98 ± 4.4ND >30 μg/ml Bmax (% live 70.9 ± 2.0 48.78 ± 10  ND indeterminate cells)

Functional activity in inhibiting bFGF-mediated/VEGF-independent (Falconet al. 2013) angiogenesis of human umbilical vein cells (HUVECs), thestandard in angiogenesis assays, was next tested (FIGS. 39A-39D).Parameters for complex formation were first measured—polygons (abilityto form a closed interconnected loop of neovessels (tube polygon), andbranch points (ability for angiogenic endothelial cells to initiatenetwork formation from a node).

After a pilot study of inhibition of angiogenesis, dose-dependentinhibition of angiogenesis experiments were performed using 6g8^(IgG4)humabl lead candidate (FIGS. 40A-40B) to obtain IC50 values to ascertainfunctional activity.

These data demonstrate that the 6g8IgG4 humab1 exhibits more robustfunctional activity than the 6g8 mumab as shown in the IC50 values(Table 4), concordant with observations for inhibition ofneutrophil-survival.

TABLE 4 6g8^(IgG4) 6g8^(IgG1) 6g8^(IgG4) humab1 humab1 humab2 6g8 mumabTable 4. Human Angiogenesis assay: complex network formation parameter =polygons IC₅₀ (ug/ml) 2.6 ± 0.2  9.1 ± 1.5 ND >30 μg/ml Bmax (%) 28 ± 4  9.7 ± 15 ND indeterminate Human Angiogenesis assay: complex networkformation parameter = branch points IC₅₀ (ug/ml) 4.3 ± 0.7 10.4 ± 2.6ND >30 μg/ml Bmax (%) 45.7 ± 7   37.2 ± 9.5 ND indeterminate

Accordingly, described herein is the successful development andcharacterization of a lead candidate for a humanized anti-DEspRmonoclonal antibody through recombinant technology with a human S228PIgG4-framework, referred to herein as “6g8^(IgG4) humab” or6g8g7-derived IgG4 humanized antibody. This humanized antibody hasrobust transient expression in HEK293 cells and has improved binding toDEspR compared to 6g8 murine monoclonal antibody on intact cells andfunctional activity, including inhibition of activated neutrophilsurvival and angiogenesis, which are two mechanisms of blood brainbarrier disruption in stroke. These results are summarized in Table 5.

TABLE 5 Table 5. Binding characteristics and effector activities ofcandidate 6g8-humabs. 6g8^(IgG4) 6g8^(IgG1) 6g8^(IgG4) humab1 humab1humab2 m6g8 ELISA (Antigenic Peptide) EC₅₀ (ug/ml) 11.7 ± 2.2 13.4 ± 2.54.8 ± 1.2 2.96 ± 0.93 Bmax (A₄₅₀)  2.62 ± 0.16 3.09 ± 0.2 1.31 ± 0.086.36 ± 0.48 Binding to DEspR on intact human cells (FACS) EC₅₀ (ug/ml) 0.64 ± 0.25  1.3 ± 0.74 ND 5.39 ± 1.84 EC₅₀ (nM) 21.1 ± 8.3 42.9 ± 24 ND 177.9 ± 60   Bmax 31.5 ± 2.6 21.6 ± 3.0 ND 35.6 ± 4.0  (% + cells)Rat activated Neutrophil survival IC₅₀ (ug/ml) 1.16 ± 0.3 6.98 ± 4.4ND >30 μg/ml Bmax 70.9 ± 2.0 48.78 ± 10   ND indeterminate (% livecells) Human Angiogenesis assay: complex network formation parameter =polygons IC₅₀ (ug/ml)  2.6 ± 0.2  9.1 ± 1.5 ND >30 μg/ml Bmax (%)  28 ±4  9.7 ± 15 ND indeterminate Human Angiogenesis assay: complex networkformation parameter = branch points IC₅₀ (ug/ml)  4.3 ± 0.7 10.4 ± 2.6ND >30 μg/ml Bmax (%) 45.7 ± 7   37.2 ± 9.5 ND indeterminate ELISA wasperformed with antigenic peptide (AP): EMKSRWNWGS (SEQ ID NO: 2);binding of AF-568-labeled mAbs to Panc1 cells was quantified by FACSwith corresponding isotype labeled antibodies as background controls;Neutrophil survival assays were performed with freshly isolated ratneutrophils. Neutrophils (50000/well) were incubated in the absence orpresence of mAbs at 37° C. × 4 hrs and live cells counted by usingTrypan blue. HUVEC-dependent angiogenesis (20,000 cells/well) wasperformed as described in the presence or absence of mAbs. Number ofpolygons and number of branch points were determined after 14 hrs ofincubation at 37° C. ND, not done, indeterminate, value greater thanupper limit of max dose used in these experiments.

Next, IC50 of anti-DEspR 6g8IgG4 humab1 inhibition of angiogenic tubelength is measured, which represents the ability of angiogenicendothelial cells to proliferate, migrate and align, and subsequentlyfuse together to form tubes (in vitro neovessels).

Further, in vitro efficacy (IC50) of the lead candidate, 6g8IgG4 humab1in inhibiting myeloperoxidase (MPO) release by activated neutrophils ismeasured. MPO is a mediator of neutrophil-mediated injury to the bloodbrain barrier during ischemic stroke.

REFERENCES

Yang X, Ambrogelly A. 2014. Enlarging the repertoire of therapeuticmonoclonal antibodies platforms: domesticating half molecule exchange toproduce stable IgG4 and IgG1 bispecific antibodies. Current Opinion inBiotechnology 30: 225-229.

Yang X, Wang F, Zhang Y, Wang L, Antonenko S, Zhang S, Zhang Y W,Tabrizifard M, Emakov G, Wiswell D, Beaumnt M, Liu L, Richardson D,Shameem M, Ambrogelly A. 2015. Comprehensive Analysis of the TherapeuticIgG4 Antibody Pembrolizumab: Hinge Modification Blocks Half MoleculeExchange In Vitro and In Vivo. J Pharm Sci 104:4002-4014.

Silva J P, Vetterlein O, Jose J, Peters S, Kirby H. 2015. The S228Pmutation prevents in vivo and in vitro IgG4 Fab-arm exchange asdemonstrated using a combination of novel quantitative immunoassays andphysiological matrix preparation. J Biol Chem 290:5462-5469.

METHODOLOGY

Each gene for heavy and light chain expression was synthesized andcloned into a mammalian expression using standard methods. Each completeconstruct was sequence verified by DNA sequencing. Plasmid DNA wasproduced for transfection into suspension HEK293 cells grown inserum-free chemically-defined medium. Antibodies from conditioned mediawere purified by binding to and elution from Protein A columns withsubsequent filtration through 0.2 μm membrane filters. Buffer exchangewas either into HEPES: 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 orPBS pH 7.4. Protein concentrations were calculated from OD280. CE-SDSanalysis of antibodies were performed using LABCHIP GXII (Perkin Elmer).

The sequences of the variable heavy and variable light chains of the6G8G7 variant antibodies are disclosed elsewhere herein as SEQ ID NOs: 6(variable heavy 1), 13 (variable heavy 2), 27 (variable light 1), 34(variable light 8), and 41 (variable light 2). The sequences of thevariable heavy and light chain of the 7C5B2 variant antibodies aredisclosed elsewhere herein as SEQ ID NO: 20 (variable heavy 2) and SEQID NO: 50 (variable light 2).

1. A method of inhibiting angiogenesis in a subject having a disease ordisorder dependent or modulated by angiogenesis, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising: (i) a pharmacuticallyaccpetable carrier and (ii) an isolated anti-DEspR antibody orantigen-binding fragment thereof that specifically binds DEspR (dualendothelin/VEGF signal peptide receptor) of SEQ ID NO: 33 comprising oneor more heavy and light chain complimentarity determining regions (CDRs)selected from the group consisting of: a. a heavy chain CDR1 having theamino acid sequence of SEQ ID NO: 7, SEQ ID NO: 14, or SEQ ID NO: 21; b.a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8, SEQID NO: 15, or SEQ ID NO: 22; c. a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 9, SEQ ID NO: 16, or SEQ ID NO: 23; d. a lightchain CDR1 having the amino acid sequence of SEQ ID NO: 28, SEQ ID NO:35, SEQ ID NO: 42, or SEQ ID NO: 51; e. a light chain CDR2 having theamino acid sequence of SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 43, orSEQ ID NO: 52; and f. a light chain CDR3 having the amino acid sequenceof SEQ ID NO: 30, SEQ ID NO: 37, SEQ ID NO: 44, or SEQ ID NO:
 53. 2. Themethod of claim 1, that is an antibody, wherein the antibody is achimeric, humanized, or composite human antibody or dual antibody. 3.The method of claim 1, that is an antigen-binding fragment thereof,wherein the antigen-binding fragment is a Fab fragment, a Fab′ fragment,a Fd fragment, a Fd′ fragment, a Fv fragment, a dAb fragment, a F(ab′)₂fragment, a single chain fragment, a diabody, or a linear antibody. 4.The method of claim 1, wherein the isolated anti-DEspR antibody orantigen-binding fragment thereof specifically binds to an epitope ofDEspR (dual endothelin/VEGF signal peptide receptor) of SEQ ID NO: 1 orSEQ ID NO:
 2. 5. The method of claim 1, wherein the isolated anti-DEspRantibody or antigen-binding fragment thereof comprises heavy and lightchain complimentarity determining regions (CDRs) selected from the groupconsisting of: a. a heavy chain CDR1 having the amino acid sequence ofSEQ ID NO: 7; a heavy chain CDR2 having the amino acid sequence of SEQID NO: 8; a heavy chain CDR3 having the amino acid sequence of SEQ IDNO: 9; a light chain CDR1 having the amino acid sequence of SEQ ID NO:28; a light chain CDR2 having the amino acid sequence of SEQ ID NO: 29;and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 30;b. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 7; aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8; a heavychain CDR3 having the amino acid sequence of SEQ ID NO: 9; a light chainCDR1 having the amino acid sequence of SEQ ID NO: 35; a light chain CDR2having the amino acid sequence of SEQ ID NO: 36; and a light chain CDR3having the amino acid sequence of SEQ ID NO: 37; c. a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 7; a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 8; a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 9; a light chain CDR1having the amino acid sequence of SEQ ID NO: 42; a light chain CDR2having the amino acid sequence of SEQ ID NO: 43; and a light chain CDR3having the amino acid sequence of SEQ ID NO: 44; d. a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 7; a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 8; a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 9; a light chain CDR1having the amino acid sequence of SEQ ID NO: 51; a light chain CDR2having the amino acid sequence of SEQ ID NO: 52; and a light chain CDR3having the amino acid sequence of SEQ ID NO: 53; e. a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 14; a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 15; a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 16; a light chain CDR1having the amino acid sequence of SEQ ID NO: 28; a light chain CDR2having the amino acid sequence of SEQ ID NO: 29; and a light chain CDR3having the amino acid sequence of SEQ ID NO: 30; f. a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 14; a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 15; a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 16; a light chain CDR1having the amino acid sequence of SEQ ID NO: 35; a light chain CDR2having the amino acid sequence of SEQ ID NO: 36; and a light chain CDR3having the amino acid sequence of SEQ ID NO: 37; g. a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 14; a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 15; a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 16; a light chain CDR1having the amino acid sequence of SEQ ID NO: 42; a light chain CDR2having the amino acid sequence of SEQ ID NO: 43; and a light chain CDR3having the amino acid sequence of SEQ ID NO: 44; h. a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 14; a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 15; a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 16; a light chain CDR1having the amino acid sequence of SEQ ID NO: 51; a light chain CDR2having the amino acid sequence of SEQ ID NO: 52; and a light chain CDR3having the amino acid sequence of SEQ ID NO: 53; i. a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 21; a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 22; a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 23; a light chain CDR1having the amino acid sequence of SEQ ID NO: 28; a light chain CDR2having the amino acid sequence of SEQ ID NO: 29; and a light chain CDR3having the amino acid sequence of SEQ ID NO: 30; j. a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 21; a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 22; a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 23; a light chain CDR1having the amino acid sequence of SEQ ID NO: 35; a light chain CDR2having the amino acid sequence of SEQ ID NO: 36; and a light chain CDR3having the amino acid sequence of SEQ ID NO: 37; k. a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 21; a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 22; a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 23; a light chain CDR1having the amino acid sequence of SEQ ID NO: 42; a light chain CDR2having the amino acid sequence of SEQ ID NO: 43; and a light chain CDR3having the amino acid sequence of SEQ ID NO: 44; and l. a heavy chainCDR1 having the amino acid sequence of SEQ ID NO: 21; a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 22; a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 23; a light chain CDR1having the amino acid sequence of SEQ ID NO: 51; a light chain CDR2having the amino acid sequence of SEQ ID NO: 52; and a light chain CDR3having the amino acid sequence of SEQ ID NO:
 53. 6. The method of claim1, wherein the antibody is a humanized antibody comprising a humanizedvariable heavy chain amino acid sequence of SEQ ID NO: 55, a humanizedvariable heavy chain IgG1 amino acid sequence of SEQ ID NO: 61, ahumanized variable heavy chain IgG4 amino acid sequence of SEQ ID NO:63, a humanized variable light chain amino acid sequence of SEQ ID NO:57, a humanized variable light chain amino acid sequence of SEQ ID NO:59, a humanized variable kappa light chain amino acid sequence of SEQ IDNO: 65, or any combination thereof.
 7. The method of claim 1, whereinthe disease or disorder dependent or modulated by angiogenesis is acancer or a tumor.
 8. The method of claim 7, wherein the disease ordisorder dependent or modulated by angiogenesis is selected from thegroup consisting of age-related macular degeneration, carotid arterydisease, diabetic retinopathy, rheumatoid arthritis, neurodegenerativedisorder, Alzheimer's disease, obesity, endometriosis, psoriasis,atherosclerosis, ocular neovascularization, neovascular glaucoma,osteoporsosis, and restenosis.
 9. The method of claim 1, wherein theisolated antibody or antigen-binding fragment thereof is a neutralizingantibody or antigen-binding fragment thereof that has at least one ofthe following functional characteristics: a. an EC50 for binding toDEspR (dual endothelin/VEGF signal peptide receptor) of SEQ ID NO: 3 of12 μg/ml or less; b. an IC50 for inhibiting activated neutriphilsurvival or human angiogenesis of 3.0 μg/ml less; or c. a K_(D) forbinding DEspR of SEQ ID NO: 3 of 2.5 μg/ml or less.
 10. A method ofinhibiting angiogenesis in a subject having a disease or disorderdependent or modulated by angiogenesis, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising: (i) apharmaceutically acceptable carrier, and (ii) an isolated antibody orantigen-binding fragment thereof capable of binding to DEspR (dualendothelin-1/VEGF signal peptide receptor) of SEQ ID NO: 3 comprisingheavy and light chain sequences comprising complementarity determiningregions (CDRs) of a heavy chain CDR1 having the amino acid sequence ofSEQ ID NO: 14; a heavy chain CDR2 having the amino acid sequence of SEQID NO: 15; a heavy chain CDR3 having the amino acid sequence of SEQ IDNO: 16; a light chain CDR1 having the amino acid sequence of SEQ ID NO:28; a light chain CDR2 having the amino acid sequence of SEQ ID NO: 29;and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 30.